Biochemistry Free Easy 1

Dedication & Credits
Biochemistry Free and Easy is dedicated to

all of the people who have inspired us. Kevin
would like to acknowledge his mentors, Dr.
E.C. Nelson from Oklahoma State University
and Dr. George Pearson from Oregon State
University. Both were instrumental in Kevins
career development and are both mentors
and lifelong friends. Indira would like to thank
her parents, who passed on to her their love
for the life sciences and nerdy jokes, through
both their DNA and their example.
On the pages that follow are photographs of
students, past and present, who have inspired
us and continue to inspire us to teach
biochemistry. Not pictured, but included in this
list are Beth Dunfield, Dr. Brian Orth, Dr. Jill
Mooney, Dr. Jeremy Gregory, Dr. Keith
Hazleton, Marsha Lampi, Dr. Tasha Ludwick,
Thomas Pitts, Varunika Bhargava, and Zebulon
Jones. The students here are a small subset of
the thousands of students who have touched
our lives and that we are grateful to have had
the chance to work with at OSU.
Dr. E.C. Nelson
Dr. George Pearson
Please note that though the photos are small, users can enlarge them by touching them on an
iPad.
Biochemistry Free and Easy 2012, 2013

Kevin Ahern & Indira Rajagopal / All rights reserved
Version 2.0
All images credited to Wikipedia have license information on the Copyright

section beginning HERE.
i
Alex Aljets
Allex Hadduck
Dr. Anahita
Fallahi
Andres Cardenas
Ashley
Meganck
Ashley Swander
Callia
Palioca
Carly Dougher
Amanda
Schwartz
Amber Bannon
Angel Garbarino
Bailey
Lindenmaier
Casey Luce &

Elyssa Ackerman
Dr. Amber Leis
Dr. Anna Hsu
Bonnie Buckingham
Cassondra Pittman
Amini Thakor
Anna Vigeland
Dr. Bory Kea
Chelsea Parker
Dr. AnneMarie Corgan
Brett Bemer
Chitra Patel
Ana Brar
Dr. April
von Allmen
Caitlin Crimp
Chris Brown
Dr. Chrissy Lamun
Chrissy Murphy
Dr. Crystal
Danya Rumore
Daniel Zollinger
David Stanley
Hammer
Dawn Gaudenti
Duy Pham
Dr. Heather
Bolstad
Deepthi Ennamuri
Denise Knaebel
Emilie Servin
Eric Brooks
Heather Hodnett
Helen Hobbs
Derek Scott
Erika Snow
Dr. Derek
Youngblood
Dr. Gautam
Mankaney
Ishan Patel
Desiree Boltz
Georgi Mitev
Dr. Jenna
Donaldson
Drew Calhoun
Hank McNett
Jenna Smith
Jennifer
Coppersmith
Dr. Jennifer
Shepard
Jessica Page with

Kevin Ahern
Julia Sobolik
Montes
Katie Brempelis
Jenny Tran, Andrea

Virvara and Valeria Ursu
Jenny Moser
Jurling
Jessica Taylor
Justin Zhang
Katie Lebold
Kaalindi
Misra
Kelsey Drewry
Jessica
Thorpe
Kara Cardwell
Dr. Kevan
Stanton
Jessica Goman
John Davidson
Kate
Bateman
Dr. Kyle Shaver
Josha
Woodward
Kate
Posposil
Dr. Laura Miller
Jessica
Kristo
Julia
Armendariz
Dr. Kathy
Tran Shaw
Leanna Mah
Lisa Weller
Dr. Mandi
Kremer
Loni Mandigo
Dr. Marie Strand
Melodie Machovina
Minhazur
Sarker
Dr. Monika
Arora Sanghavi
Loren Brown
Mark Hall
Michelle Buchanan
Dr. Nancy
Jade Lee
Ngan Nguyen
Mallori
Jirikovic
Lotti Alvord
Matt Lewis and

Christine Schneider
Michelle
Nguyen
Nick Lowery
Megan Cook
Dr. Michinao
Hashimoto
Nicolette
ODonnell
Meghan
Krueger
Dr. Minh Ho
Dr. Nima
Motamedi
Omar Rachdi
Robbie Lamb
Sarah Ferrer & Emily

Pickering
Stephanie
McGregor
Paul Jones and

Hannah Raines
Oresta Tolmach
Robyn Ikehara
Murakawa
Sarah Lindsley
Steve Davee
Ryan Derrah
Dr. Sathya
Sriram
Steven Craig
Brantley
Rachel
Aazzerah
Rakan Khaki
Raviteja Madhira
Sam Schuberg
Dr. Sara Haidar
Sarah and
Michael Layoun
Shannon Go
Sydney
Radding
Dr. Shawn
Werner
Shayna
Rogers
Dr. Tara Humphrey
Stephanie
Duckett
Tari Tan
Theresa & Maria Nguyen
Tony
Rianprakaisang
Wren Patton
Tristan
Wagner
TJ Brodeur
Thi Nguyen, Justin Biel &

Margo Roemeling
Dr. Valerie
Bomben
Valerie Mullen
Yesenia Correa
Dr. Tom Sharpton
Vihangi Hindagolla
Zoe Gombart
David Simmons, Liz

Bacon, Barbara & Neal
Gladstone, and Tim
Karplus brought the
Metabolic Melodies to
Credits
life with their delightful

recordings that neither of
We are indebted to several

people for their assistance in
putting together this book.
Jana Rade is a generous,
wonderfully talented artist who
created the beautiful cover in
the spirit of this book - for
FREE, which was great because we didnt have much of a
budget. Her graphic design business is impact studios and she
can be reached at jrade@impactstudiosonline.com. Please
support Janas Web site at www.impactstudiosonline.com
Martha Jane Baker, an extraordinarily talented young Oregon
us could ever dream of

David Simmons
doing. David is currently a
medical student at Oregon Health

and Science University and will
soon be Dr. David Simmons.
Barbara and Neal Gladstone are
wonderful friends of ours who
can be reached through their
musical Web site www.nealgladstone.com
Indira and Kevin are faculty
members in the Department of
State University student who created, or oversaw the creation of,
Biochemistry and Biophysics (link
all of the original artwork here. Please support her Web site at the
HERE) at Oregon State University
following URL - www.mjbakerartist.com. She was assisted by
(link HERE) where we teach
Alyssa Hughes. Student wages for Martha and Alyssa were partly
biochemistry, molecular biology and Honors biology to hundreds
provided by a faculty grant to Kevin from the L.L. Stewart fund
of terrific students each year and write verses and songs about
Liz Bacon
supporting faculty eorts that improve teaching at OSU.

viii
biochemistry called Metabolic Melodies,
We have a Web site full of Metabolic Melodies
many of which were used here.
and Limericks that you may enjoy. In the spirit

of this book, everything there is free too. The
We decided when we created this book
URL is
that information should be as freely

available as possible. We appreciate your
http://www.davincipress.com/
support (financial or otherwise) to say
metabmelodies.html
thanks for our eorts. Ways to do this
Kevin has also released a book of limericks titled
include buying a calendar (HERE) or the
A Limerick A Day For A Year More
The Splendidest Ever Metabolic

Melodies songbook (HERE), registering for our
e-campus
information about that HERE.
classes at Oregon State University (HERE, HERE, HERE, and
He also a few YouTube music videos he is proud of. They include
HERE), and/or sending us a message telling us how youve used
an anti-smoking video called I Lost a Lung (from smoking
the book. You can also join and LIKE our Facebook page for
Biochemistry Free and Easy HERE.
Our email addresses are
kevin@davincipress.com
indira@davincipress.com
You can view our biochemistry courses as
Camels) and is to the tune of I Left My Heart in San

Francisco The link is HERE. The Oregon rain gets
noted in Let It Rain (to the tune of Let It Snow)
HERE and Around the Nucleus (to the tune of
Across the Universe) is HERE. Last, Kevin has a
series of video workshops of interest to pre-medical
students. These can be accessed HERE
follows below:
BB 350 - HERE / Syllabus HERE
BB 450/550 - HERE / Syllabus HERE
BB 451/551 - HERE / Syllabus HERE
BB 100 - HERE / Syllabus HERE
ix
Chapter 1
Cells, Water,
and Buers
Welcome to our Biochemistry text. Were glad
youre here. In this chapter we introduce the
subject and talk about the scientific aspects of the
most important and most abundant liquid on the
face of the Earth - water.
Cells, Water, and Buers

Introduction
Cells: The Bio of Biochemistry
Introduction
Welcome to Biochemistry Free and Easy. As biochemistry instructors, we are
Water, Water Everywhere
always delighted when anyone shows an interest in our favorite subject. Helping
Buers Keep the Cellular Environment Stable
students understand and enjoy biochemistry is our motivation for writing this book.
Henderson-Hasselbalch
It is not our intention to provide a comprehensive account of the chemical basis of

life. Instead, we have tried to help students understand this fascinating subject by
focusing on some key topics and concepts. This pared-down approach can be
helpful for novices who might otherwise lose sight of important organizing principles
in a sea of detail. The electronic format has also allowed us to provide multimedia
links, in the form of video lectures and biochemistry songs to help students learn the
subject. Best of all, this format makes it practical for us to distribute the book at no
cost to anyone who wishes to learn basic biochemistry.
Biochemistry is a relatively young science, but the rate of its expansion has been
truly impressive. This rapid pace of
discoveries, which shows no signs of
slowing, is reflected in the steady
increase in the size of biochemistry text
books, most of which top a thousand
pages and undergo revisions every
On these pages our aims to extract

Knowledge out of a mountain of facts
Missing excessive data
Will not really matta
If our students can learn and relax
couple of years to incorporate new

11
findings. These full-scale

texts oer an enormous
amount of information and
serve as invaluable
resources. Those who
need the greater level of
detail and broader
coverage that these books
provide have many
choices available in any
good bookstore.
As certified (some might
say, certifiable)
biochemistry nerds and
unrepentant lovers of
corny jokes, we firmly
Biochemistry, Biochemistry
To the tune of Oh Christmas Tree
Biochemistry Biochemistry
I wish that I were wiser
I feel Im in way oer my head
I need a new advisor
I promise I would not complain

If I could store them in my brain
My courses really shouldnt be

Such metabolic misery
Im truly in a panic
Your mechanisms murder me
I should have learned organic
Reactions make me shiver
Theyre in my heart and in my lungs
Theyre even in my liver
For all I have to memorize

I ought to win the Nobel Prize.
believe that students can

have fun while learning the
subject. Toward this end,
we have sprinkled each
Biochemistry, Biochemistry
chapter with rhymes and

songs that we hope will
have you learning
Recorded by Tim Karplus.

Lyrics by Kevin Ahern
biochemistry happily. The

format of the book as available for the iPad, allows readers to
to each topic, listen to the songs in the book, like the one
click on figures to enlarge them, watch video lectures relevant
above, and link out to the internet to find more information

12
simply by clicking on any term. If you are using a PDF

version of this book, you will still be able to use the links to
the video lectures. Also, though you cannot listen to the
songs by clicking on them in the PDF version, you can
download them HERE. We hope you find these features
useful and that they help you learn biochemistry.
Cells: The Bio of Biochemistry

Biochemistry happens inside organisms and possibly, the
most obvious thing about living organisms is their
astounding diversity. If living things are so varied, it seems
reasonable to ask whether their chemistry is, too.
The invention of the microscope opened up a whole new
world of microscopic organisms while also providing the
Onion cells
first clue that living organisms had something in common-
from Wikipedia
all living things are made up of cells. Some cells are lone
rangers in the form of unicellular entities, such as bacteria
that lack such internal compartmentation, the prokaryotes. Some
and some protists. Cells are also the building blocks of more
eukaryotes, such as yeast, are unicellular, while others, including
complex organisms (like humans, wombats, and turnips).
animals and plants are multicellular. The prokaryotes may be
As increasingly powerful microscopes

became available, it was possible to discern
that all cells fell into one of two types- those
with a nucleus and other sub-cellular
compartments like mitochondria and
lysosomes, termed eukaryotes, and those
divided into two very broad categories, the bacteria and the
archaeans.
Excuse me for feeling superia
To the life forms we call the bacteria
Students know very well
There are no organelles
To be found in their tiny interia
One can find living cells almost everywhere on

earth - in thermal vents on the ocean floor, on
the surface of your tongue and even in the
frozen wastes of the Antarctic. Some cells may
13
the evolutionary spectrum. Where cells dier significantly in

processes/reactions, we will note these dierences.
Water, Water,
Everywhere
Vital for life, water is by far
the most abundant
Click HERE for Kevins

Introductory Lecture
on YouTube
component of every cell. To

understand life, we must, therefore, understand the basics of
water, because everything that happens in cells, even reactions
buried deep inside enzymes, away from water, is influenced by
Extremophiles
waters chemistry.
from Wikipedia
have even survived over two years on the moon1. Yet,

despite their diversity of appearance, habitat, and genetic
composition, cells are not as dierent from each other as
you might expect. At the biochemical level, it turns out that
all cells are more alike than they are dierent. A great
simplifying feature of biochemistry is that many of the
reactions are universal, occurring in all cells. For example,
most bacteria process glucose in the same 10-step
pathway that plant, animal, and fungal cells do. The
genetic code that specifies the amino acids encoded by a
nucleic acid sequence is interpreted almost identically by
all living cells, as well. Thus, the biochemical spectrum of
life is (mercifully) not nearly as broad or as complicated as
from Wikipedia
14
We measure the proton concentration of a solution with pH,

which we define as the negative log of the proton concentration.
pH = -Log[H+]
If the proton concentration, [H+]= 10-7 M, then the pH is 7. We
could just as easily measure the hydroxide concentration with the
pOH by the parallel equation,
pOH = -Log[OH-]
In pure water, dissociation of a proton from it creates a hydroxide,
We start with simple properties. The molecule has a sort of wide

V shape (the H-O-H angle is 104) with uneven sharing of
electrons between the oxygen and the hydrogens. The
hydrogens, as a result, are described as having a partial positive
charge and the oxygen has a partial negative charge. These tiny
partial charges allow the formation of what are described as
hydrogen bonds, which occur when the partial positive charge of
so the pOH of pure water is 7, as well. This also means that

pH + pOH = 14
Now, because protons and hydroxides can combine to form
water, a large amount of one will cause there to be a small
amount of the other. Why is this the case? In simple terms, if I
dump 0.1 moles of H+ into a pure water solution, the high proton
one atom is attracted to the partial negative of another. In water,

that means the hydrogen of one water molecule will be attracted
Why do we care about pH? Because biological
to the oxygen of another. Hydrogen bonds play essential roles in
molecules can, in some cases, be exquisitely sensitive
proteins, DNA, and RNA, as well, as we shall see.
to changes in it. As the pH of a solution changes, the
Buers Keep the Cellular Environment

Stable
Water can ionize to a slight extent (10-7 M - about 6 molecules per
charges of molecules in the solution can change, as

you will see. Changing charges on biological
molecules, especially proteins, can drastically aect
how they work and even whether they work at all.
100 million of pure water) to form H+ (proton) and OH- (hydroxide).

15
16
concentration will react with the relatively small
Clearly, weak acids are very dierent from strong
amount of hydroxides to create water, thus
Buer Lecture
reducing hydroxides. Similarly, if I dump excess
acids. Weak bases behave similarly, except that

they accept protons, rather than donate them.
on YouTube
hydroxide (as NaOH, for example) into pure water,
You may wonder why we care about weak acids.
the proton concentration falls for the same
You may never have thought much of weak acids
reason.
when you were in General Chemistry. Your instructor described
Chemists use the term acid to refer to a substance which has
them as buers and you probably dutifully memorized the fact
protons that can dissociate (come o) when dissolved in water.
that buers are substances that resist change in pH without
They use the term base to refer to a substance that can absorb
really learning what it meant. We will not allow that to happen
protons when dissolved in water. Both acids and bases come in
here.
strong and weak forms. (Examples of weak acids are shown on

the previous page.) Strong acids, such as HCl, dissociate
completely in water. If we add 0.1 moles of HCl to a solution to
make a liter, it will have 0.1 moles of H+ and 0.1 moles of Cl-.
There will be no remaining HCl when this happens. A strong base
like NaOH also dissociates completely into Na+ and OH-.
Weak acids are critical for life because their anity for protons
causes them to behave like a UPS. Were not referring to the
UPS that is the United Parcel Service, but instead, to the
encased battery backup systems for computers called
Uninterruptible Power Supplies that kick on to keep a computer
running during a power failure. Your laptop battery is a UPS, for
Weak acids and bases dier from their strong counterparts.
example. We can think of weak acids as Uninterruptible Proton
When you put one mole of acetic acid (HAc) into pure water, only
Suppliers within certain pH ranges, providing (or absorbing)
On this chemical fact we must dwell

H-A-Cs just not like H-C-L
It always negotiates
Before it dissociates
To bid every proton farewell
about 4 in 1000 HAc
protons as needed.
molecules dissociate
Weak acids thus
into
H+
and
Ac-.
Thus,
help to keep the
H+
if I start with 1000
concentration (and
HAc, I will end up
thus the pH) of the
with 996 HAc and 4
solution they are in
each of H+ and Ac-.
relatively constant.
I confess I am pleased to possess

In my buers a strong UPS
Giving Hs when needed
Grabbing same when exceeded
So my cells dont get proton distressed
17
Consider the acetic acid (acetate) system. Here is what happens

when HAc dissociates
HAc <=> H+ + AcAs noted, about 4 in 1000 HAc molecules come apart. However,
what if one started adding hydroxyl ions (by adding a strong base
like NaOH) to the solution with the HAc in it? As the added OHions reacted with the H+ ions to make water, the concentration of
H+ ions would go down and the pH would go up. However, in
contrast to the situation with a solution of pure water, there is a
backup source of H+ available in the form of HAc. Here is where
the UPS function kicks in. As protons are taken away by the
added hydroxyl ions (making water), they are partly replaced by
protons from the HAc. This is why a weak acid is a buer. It
resists changes in pH by releasing protons to compensate for
those used up in reacting with the hydroxyl ions.
Henderson-Hasselbalch
It is useful to be able to predict the response of the HAc system
to changes in H+ concentration. The Henderson-Hasselbalch
equation (click HERE for an interactive module) defines the
relationship between pH and the ratio of
Ac-
and HAc. It is as
follows
pH = pKa + log ([Ac-]/[HAc])
An example buffer system - acetic acid/acetate (most commonly

called an acetate buffer). Note that near pH 4.76, addition of
OH- results in only small pH changes. This is what a buffer
does - resists changes in pH over certain ranges.
This simple equation defines the relationship between the pH of a
solution and the ratio of Ac- and HAc in it. The new term, called
the pKa, is defined as
pKa = -Log Ka, just as pH = -Log [H+]
The Ka is the acid dissociation constant and is a measure of the
strength of an acid. For a general acid, HA, which dissociates as
18
Please note that pKa is a constant for a given acid. The pKa for
Why do we care about buers? Buers help to keep the pH of
a solution from changing much, even when protons are added
to it or removed from it. When you exercise, your muscles
produce protons, which get dumped into the blood. If a buer
were not present, the pH of the blood would change drastically
and you would likely die, since the acidification of your blood
would denature/inactivate most of your enzymes
HA <=> H+ + A-,
acetic acid is 4.76. By comparison, the pKa for formic acid is

3.75. Formic acid is therefore1a stronger acid than acetic acid. A
stronger acid will have more protons dissociated at a given pH
than a weaker acid.
Now, how does this translate into stabilizing pH? The previous
figure shows a titration curve. In this curve, the titration begins
with the conditions at the lower left (very low pH). At a this pH,
the HAc form predominates, but as more and more OH- is added
Ka = [H+][A-]/[HA]
Thus, the stronger the acid, the more protons that will dissociate
from it and the larger the value its Ka will have. Large values of Ka
(moving to the right), the pH goes up, the amount of Ac- goes up
and (correspondingly), the amount of HAc goes down. Notice
that the curve flattens near the pKa (4.76).
translate to lower values of pKa. As a result, the lower the pKa
What this tells us is that the pH is not changing much (not going
value is for a given acid, the stronger the acid is.
up as fast) as it did earlier when the same amount of hydroxide

was added. The system is resisting a change in pH (not stopping
the change, but slowing it) in the region of about one pH unit
above and one pH unit below the pKa. Thus, the buering region
of the acetic acid/acetate buer is from about 3.76 to 5.76. It is
maximally strong at a pH of 4.76.
Now it starts
to become
apparent
how the
buer works.
Why does the pKa give the pH of the maximum

buering capacity? From the HendersonHasselbalch equation, when pH = pKa, the log
term (log{[A-]/[HA]) must be zero. For the log
term to be zero, [A-] must equal [HA].
HA can
19
Henderson Hasselbalch
To the tune of "My Country 'Tis of Thee"
Henderson Hasselbalch
You put my brain in shock
Oh woe is me
The pKas can make
Me lie in bed awake
They give me really bad headaches
Oh hear my plea
Salt - acid RA-ti-os

Help keep the pH froze
By buf-fer-ING
They show tenacity
Complete audacity
If used within capacity
To maintain things
I know when Hs fly

A buffer will defy
Them actively
Those protons cannot waltz
When they get bound to salts
With this the change in pH halts
All praise to thee
Thus now that Ive addressed

This topic for the test
Ive got know-how
The pH I can say
Equals the pKa
In sum with log of S oer A
I know it now
donate protons when extras are needed (such as when

OH- is added to the solution. Similarly, A- can accept
protons when extra H+ are added to the solution (adding
HCl, for example). The maximum ability to donate or
accept protons comes when
[A-] = [HA]
To understand how well a buer protects against
changes in pH, consider the eect of adding .01 moles of
HCl to 1.0 liter of pure water (no volume change) at pH 7,
compared to adding it to 1.0 liter of a 1M acetate buer
at pH 4.76. Since HCl completely dissociates, in 0.01M
(10-2 M) HCl you will have 0.01M H+. For the pure water,
the pH drops from 7.0 down to 2.0 (pH = -log(0.01M)).
By contrast, the acetate buers pH is 4.74. Thus, the
pure water solution sees its pH fall from 7 to 2 (5 pH
units), whereas the buered solution saw its pH drop
from 4.76 to 4.74 (0.02 pH units). Clearly, the buer
minimizes the impact of the added protons compared to
the pure water.

It is important to note that buers have capacities limited
by their concentration. Lets imagine that in the previous
Recorded by David Simmons

Lyrics by Kevin Ahern and Indira Rajagopal
paragraph, we had added the 0.01 moles HCl to an

acetate buer that had a concentration of 0.01M and
equal amounts of Ac- and HAc. When we try to do the
20
math in parallel to the previous calculation, we see that there

are 0.01M protons, but only 0.005M A- to absorb them. We
could imagine that 0.005M of the protons would be
absorbed, but that would still leave 0.005M of protons
unbuered. Thus, the pH of this solution would be
approximately
pH = It surely cannot get much tougher
Than exceeding the range of your buer
All those excess H-plusses
Will raise a big ruckus
As the falling pH makes you suer
log(0.005M)
= 2.30
Exceeding
buering
capacity
dropped the
pH significantly compared to adding the same amount of

protons to a 1M acetate buer. Consequently, when
considering buers, it is important to recognize that their
concentration sets their limits. Another limit is the pH range in
which one hopes to control proton concentration.
Now, what happens if a molecule has two (or more) ionizable
groups? It turns out, not surprisingly, that each group will have its
own pKa and, as a consequence, will tend to ionize at dierent
pH values. The figure above right shows the titration curve for a
simple amino acid, alanine. Note that instead of a single
Titration Plot for Alanine
each centered on the respective pKa values for the carboxyl

group and the amino group.
If we think about alanine, it can have three possible charges: +1
(alpha carboxyl group and alpha amino group each has a proton),
0 (alpha carboxyl group missing a proton and alpha amino group
has a proton) and -1 (alpha carboxyl group and alpha amino
group each lacking a proton).
flattening of the curve, as was seen for acetic acid, alanine
How does one predict the charge at a given pH for an amino
displays two such regions. These are individual buering regions,
acid? A good rule of thumb for estimating charge is that if the pH

21
is more than one unit below the pKa for a
Around the Nucleus
group (carboxyl or amino), the proton is on. If
To the tune of Across the Universe
the pH is more than one unit above the pKa for

the group, the proton is o. If the pH is NOT
DNA gets spooled like balls of yarn

Within the chromosomes
Unwinding when its duplicated there
Around the nucleus
Primase sets down RNA

To pave the path for DNA
Across a replication fork
Complementarit-y rules (ahhhhhh)

DNA Pol-y-mer-ase
Synthesizing DNAs
RNA Pol-y-mer-ase
Making all the RNAs
Helicases split the strands

In front of replication forks
To make templates accessible
Around the nucleus
Complementary bases
Match the bonds of H and hold the
strands
Together till theyre pulled apart
more than one or less than one pH unit from the

pKa, this simple assumption will not work.
Further, it is important to recognize that these
rules of thumb are estimates only. The pI (pH at
which the charge of a molecule is zero) is an
exact value calculated as the average of the two
pKa values on either side of the zero region. It is
calculated at the average of the two pKa values
around the point where the charge of the
molecule is zero.
Reference
1. http://www.lpi.usra.edu/lunar/missions/apollo/
apollo_12/experiments/surveyor/
Around the nucleus

Hydrogen bonding fuels (ahhhhhhhhh)
Tiny alpha helix bands
Folding for the cells demands
Beta sheets comprised of strands
Meeting all the cells demands
Exons link majestically all guided

By a master plan encoded in the cells
genome
Thats buried deep inside of me
Countless combinations of the codons
Bring diversity to life evolving on and on
Around the nucleus
Complexes rule the world (ahhhhhhhh)

Ribosomes and spliceosomes
Transforming the cells genomes
Ribosomes and spliceosomes
Builders of the proteomes
YouTube video HERE

22
Click on the NEXT button below to continue.
Interactive 1.1 An interactive module involving the Henderson-Hasselbalch equation
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
23
Chapter 2
Energy
In this chapter we discuss the most important factor

necessary for life - energy.
Energy
Introduction
Oxidative Energy
Introduction
Living organisms are made up of cells, and cells contain many biochemical
Oxidation vs. Reduction
components such as proteins, lipids, and carbohydrates. But, living cells are not
Energy Coupling
random collections of these molecules. They are extraordinarily organized or
Entropy and Energy

Gibbs Free Energy
Cellular Phosphorylations
"ordered". By contrast, in the nonliving world, there is a universal tendency to

increasing disorder. Maintaining and creating order in cells takes the input of
energy.
Without energy, life is not possible. It is therefore important that we consider
Substrate Level Phosphorylation
energy first in our attempt to understand biochemistry. Where does energy come
Oxidative Phosphorylation
from? Photosynthetic organisms can capture energy from the sun, converting it to
ATP Synthase
chemical forms usable by cells. Heterotrophic organisms like ourselves get our
Photophosphorylation
Chloroplasts vs. Mitochondria
energy from the food we eat. How do we extract the energy from the food we eat?
Oxidative Energy
Energy Eciency
The primary mechanism used by non-photosynthetic organisms to obtain energy
Metabolic Controls
is oxidation and carbon is the most commonly oxidized energy source. The
Molecular Battery Backups
energy released during the oxidative steps is captured in ATP and can be used
later for energy coupling (see below). The more reduced a carbon atom is, the
more energy can be realized from its oxidation. Conversely, the more oxidized a
carbon atom is, the more energy it takes to reduce it.
25
of this that we use fat as our primary energy storage material.
Oxidation vs. Reduction in Metabolism

Biochemical processes that break things down from larger to
smaller are called catabolic processes. Catabolic processes are
often oxidative in nature and energy releasing. Some, but not all
of that energy is captured as ATP. If not all of the energy is
captured as ATP, what happens to the rest of it? The answer is
simple. It is released as heat
and it is for this reason that we
Click HERE and HERE for
get hot when we exercise. By
Kevins Metabolic Strategies
contrast, synthesizing large
Lectures on YouTube
In this series, the most reduced form of carbon is on the left. The
molecules from smaller ones
energy of oxidation of each form is shown above it. Fatty acids
(for example, making proteins
are more reduced overall than sugars. This can also be seen by
from amino acids) is referred to as anabolism. Anabolic
their formulas.
processes are often reductive in nature and require energy input.
Palmitic acid = C16H34O2
By themselves, they would not occur, as they are reversing
Glucose = C6H12O6
Palmitic acid only contains two oxygens per
sixteen carbons, whereas glucose has six
oxygen atoms per six carbons.
Consequently, when palmitic acid is fully
oxidized, it generates more ATP per carbon
(128/16) than glucose (38/6). It is because
Metabolic Energy Flow

26
reaction. The energy needed

to drive reactions is
harvested in very controlled
conditions in the confines of
an enzyme. This involves a
process called coupling. In
coupled reactions, an
enzyme binds both a high
energy molecule (usually
ATP) and the other
molecule(s) involved in the
oxidation and decreasing entropy (making many small things into

a larger one). To overcome this energy barrier, cells must
expend energy. For example, if one wishes to reduce CO2 to
carbohydrate, energy must be used to do so. Plants do this
during the dark reactions of photosynthesis. The energy source
reaction. Hydrolysis of ATP

provides energy for the
enzyme to stimulate the reaction on the other substance(s).
Hexokinase, for example, catalyzes the phosphorylation of
glucose to form glucose-6-phosphate. In the absence of ATP, the
reaction has a fairly
for the reduction is ultimately the sun. The electrons for the
positive G (described
reduction ultimately come from water, and the CO2 comes from
later), but hydrolysis of
the atmosphere and gets incorporated into a sugar.
ATP provides excess

energy, giving the coupled
Energy Coupling
The addition of phosphate to a sugar is a common reaction that
reaction a fairly negative

G value.
One reason we need ATP

Is the high cost of living, you see
Cause the chaotic entity
Known as the entropy
Requires cells to burn energy
occurs in a cell. By itself, this process is not very energetically

favorable (that is, it needs an input of energy to occur). Cells
overcome this energy obstacle by using ATP to drive the
27
suit. Throw the deck into the air, letting the
Entropy and
Energy
cards scatter. When you pick them up, they will

be more disordered than when they started.
Most students who have had
However, if you spend a few minutes (and
some chemistry know about
expend a bit of energy), you can reorganize the
the principle of the Second
same deck back to its previous, organized
Law of Thermodynamics with
state. If entropy always increased everywhere,
respect to increasing disorder
you could not do this. However, with the input
of a system. Cells are very
of energy, you overcame the disorder. The cost
organized or ordered
of fighting disorder is energy.
structures, leading some to

mistakenly conclude that life
There are, of course, other reasons that
somehow violates the second
organisms need energy. Muscular contraction,
law. In fact, that notion is
synthesis of molecules, neurotransmission,
incorrect. The second law
signaling, thermoregulation, and subcellular
doesnt say that entropy
movements are examples. Where does this
always increases, just that,
energy come from? The currencies of energy
left alone, it tends to do so, in
are generally high-energy phosphate-containing
an isolated system. Cells are
molecules. ATP is the best known and most
not isolated systems, in that
abundant, but GTP is also an important energy
they obtain energy, either
Anabolism and Catabolism
from the sun, if they are

autotrophic, or food, if they are heterotrophic.
To counter the universal tendency towards disorder on a local
scale requires energy. As an example, take a fresh deck of cards
which is neatly aligned with Ace-King-Queen . . . . 4,3,2 for each
source (required for protein synthesis). CTP is

involved in synthesis of glycerophospholipids
and UTP is used for synthesis of glycogen. In each of these
cases, the energy is in the form of potential chemical energy
stored in the multi-phosphate bonds. Hydrolyzing those bonds
releases the energy in them.
28
Of the triphosphates, ATP is the

primary energy source, acting to
facilitate the synthesis of the
others by action of the enzyme
NDPK. ATP is made by three
distinct types of phosphorylation
oxidative phosphorylation (in
mitochondria),
photophosphorylation (in
chloroplasts of plants), and
substrate level phosphorylation (in
enzymatically catalyzed reactions).
Oh Delta G
To the tune of Danny Boy
Oh Delta G - the change in Gibbs free energy

Can tell us if a process will advance
'Cause if the value's less than naught it translates that
Reverse reactions haven't got a chance
But when the sign is plus it is the opposite
And then the backwards happens all the time
A factor is the standard Gibbs free energy
So don't forget about the Delta G naught prime
Wikipedia defines Gibbs free energy as a

thermodynamic potential that measures
the "useful" or process-initiating work
obtainable from an isothermal, isobaric
thermodynamic system, and further
points out that it is themaximum
amount of non-expansion work that can
be extracted from aclosed system; this
maximum can be attained only in
acompletely reversible process.
Mathematically, the Gibbs free energy is
given as
Gibbs Free Energy

Most of the time, ATP is the
G = H TS
O
where H is the enthalpy, T is the
storage battery of cells (See also
temperature in Kelvin, and S is the
Molecular Battery Backups for

Muscles below). In order to
understand how energy is
captured, we must first
Recording by Tim Karplus

entropy.
At standard temperature and pressure,
every system seeks to achieve a
understand Gibbs free energy and in doing so, we begin to see
minimum of free energy. Thus, increasing entropy will reduce
the role of energy in determining the directions chemical reactions
Gibbs free energy. Similarly, if excess heat is available (reducing
take.
the enthalpy), the free energy can also be reduced. Cells must
work within the laws of thermodynamics, as noted, so all of their
29
biochemical reactions, too, have limitations. Now we shall

consider energy in the cell. The change in Gibbs free energy (G)
for a reaction is crucial, for it, and it alone, determines whether or
not a reaction goes forward.
G = H TS,
There are three cases
G = G + RTln({Products}/{Reactants})
For most biological systems, the temperature, T, is a constant for
a given reaction. Since G is also a constant for a given
reaction, the G is
changed almost
exclusively as the
ratio of {Products}/
G < 0 - the reaction proceeds as written
{Reactants}
G = 0 - the reaction is at equilibrium
changes. If one
G > 0 - the reaction runs in reverse

For a reaction aA <=> bB (where a and b are integers and A
and B are molecules) at pH 7, G can be determined by the
following equation,
starts out at
When reactions have product largesse

They will act to address the excess
Henry Le Chatelier
Showed conversions the way
To suppress the excess by redress
standard
conditions, where everything except protons is at 1M, the
RTln({Products}/{Reactants}) term is zero, so the G term
determines the direction the reaction will take. This is why people
G = G + RTln([B]b/[A]a)
For multiple substrate reactions, such as aA + cC <=> bB + dD
G = G + RTln{([B]b[D]d)/([A]a[C]c)}
say that a negative G indicates an energetically favorable

reaction, whereas a positive G corresponds to an unfavorable
one.
Increasing the ratio of {Products}/{Reactants} causes the value of
The G term is called the change in Standard Gibbs Free
the natural log (ln) term to become more positive (less negative),
energy, which is the change in energy that occurs when all of the
thus making the value of G more positive. Conversely, as the
products and reactants are at standard conditions and the pH is
ratio of {Products}/{Reactants} decreases, the value of the natural
7.0. It is a constant for a given reaction.
log term becomes less positive (more negative), thus making the
value of G more negative.
In simple terms, if we collect all of the terms of the numerator

together and call them {Products} and all of the terms of the
Intuitively, this makes sense and is consistent with Le Chateliers
denominator together and call them {Reactants},
Principle a system responds to stress by acting to alleviate the

30
stress. If we examine the G for a reaction in a closed system,
weight in triphosphates. Since triphosphates are the currency
we see that it will always move to a value of zero (equilibrium), no
that meet immediate needs of the cell, it is important to
matter whether it starts with a positive or negative value.
understand how triphosphates are made. There are three
Another type of free energy available to cells is that generated by

electrical potential. For example, mitochondria and chloroplasts
partly use Coulombic energy (based on charge) from a proton
gradient across their membranes to provide the necessary energy
for the synthesis of ATP. Similar energies drive the transmission
of nerve signals (dierential distribution of sodium and potassium)
and the movement of some molecules in secondary active
transport processes across membranes (e.g.,
H+
dierential
driving the movement of lactose). From the Gibbs free energy
phosphorylation mechanisms 1) substrate level; 2) oxidative;

and 3) photophosphorylation. We consider them here individually.
Substrate Level Phosphorylation

The easiest type of phosphorylation to understand is that which
occurs at the substrate level. This type of phosphorylation
involves the direct synthesis of ATP from ADP and a reactive
intermediate, typically a high energy phosphate-containing
molecule. Substrate level phosphorylation is a relatively minor
change equation,
G = H TS
it should be noted that an increase in entropy will help contribute
to a decrease in G. This happens, for example when a large
molecule is being broken into smaller pieces or when the
rearrangement of a molecule increases the disorder of molecules
around it. The latter situation arises in the hydrophobic eect,
which helps drive the folding of proteins.
Cellular Phosphorylations
Formation of triphosphates is essential to meet the cells
immediate energy needs for synthesis, motion, and signaling. In
a given day, an average human being uses more than their body
31
contributor to the total synthesis of triphosphates by cells. An
phosphofructokinase, of glycolysis, that will catalyze reactions to
example substrate phosphorylation comes from glycolysis.
give the cell additional, needed energy.
Phosphoenolpyruvate (PEP) + ADP <=> Pyuvate + ATP
Electron Transport / Oxidative Phosphorylation
This reaction has a very negative G (-31.4 kJ/mol), indicating
Mitocohondria are called the power plants of the cell because
that the PEP contains more energy than ATP, thus energetically
most of a cells ATP is
favoring ATPs synthesis. Other triphosphates can be made by
produced there, in a process
substrate level phosphorylation, as well. For example, GTP can
referred to as oxidative
for Kevins Energy Lectures
be synthesized by the following citric acid cycle reaction
phosphorylation. The
on YouTube
Click HERE, HERE, and HERE
mechanism by which ATP is

Succinyl-CoA + GDP + Pi <=> Succinate + GTP + CoA-SH
Triphosphates can be interchanged readily in substrate level
phosphorylations catalyzed by the enzyme Nucleoside
made in oxidative
phosphorylation is one of the most interesting processes in all of
biology. It has three primary considerations. The first is electrical
Diphosphate Kinase (NDPK). A generalized form of the reactions

catalyzed by this enzyme is as follows:
XTP + YDP <=> XDP + YTP
Where X = adenosine, cytidine, uridine, thymidine, or guanosine
and Y can be any of these as well. Last, an unusual way of
synthesizing ATP by substrate level phosphorylation is that
catalyzed by adenylate kinase
2 ADP <=> ATP + AMP
This reaction is an important means of generating ATP when the
cell doesnt have other sources of energy. The accumulation of
AMP resulting from this reaction activates enzymes, such as
A Mitochondrial Cross-section
32
Im a Little Mitochondrion
To the tune of Im a Lumberjack
electrons move from one complex to the next, not unlike the way
they might move through an electrical circuit.
The next consideration arises as a secondary phenomenon.
I'm a little mitochondrion

Who gives you energy
I use my proton gradient
To make the ATPs
He's a little mitochondrion

Who gives us energy
He uses proton gradients
To make some ATPs
Electrons flow through Complex II

To traffic cop Co-Q
Whenever they arrive there in
An FADH-two
Electrons flow through Complex II

To traffic cop Co-Q
Whenever they arrive there in
An FADH-two
Tightly coupled is my state

Unless I get a hole
Created in my membrane by
Some di-ni-tro-phe-nol
Yes tightly coupled is his state

Unless he gets a hole
Created in his membrane by
Some di-ni-tro-phenol
Both rotenone and cyanide

Stop my electron flow
And halt the calculation of
My "P" to "O" ratio
Both rotenone and cyanide

Stop his electron flow
And halt the calculation of
His "P" to "O" ratio
When electrons pass through complexes I, III, and IV, protons are
moved from the mitochondrial matrix (inside of mitochondrion)
and deposited in the intermembrane space (between the inner
and outer membranes of the mitochondrion). The eect of this
redistribution is to increase the electrical and chemical potential
across the membrane. Students may think of the process as
charging the battery.
Just like a charged battery, the potential arising from the proton
dierential across the membrane can be used to do things. This
is the third consideration. In the mitochondrion, the thing that
the proton gradient does is create ATP from ADP and Pi
(inorganic phosphate). This process requires energy and is
accomplished by movement of protons through a protein
complex in the inner mitochondrial membrane. The protein
complex is an enzyme that has several names, including

Complex V, PTAS (Proton Translocating ATP Synthase), and ATP
Synthase. Central to its function is the movement of protons

through it (from outside back into the matrix). Protons will only
move through ATP Synthase if their concentration is greater
electrons from reduced energy carriers, such as NADH and
outside the inner membrane than in the matrix.
FADH2, enter an electron transport system via protein complexes

containing iron. As seen in the figure on the following page,
In summary, the electron transport system charges the battery for

oxidative phosphorylation by pumping protons out of the
33
In ATP Synthase, the spinning component is the

membrane portion (c ring) of the F0 stalk. The c ring
proteins are linked to the gamma-epsilon stalk, which
projects into the F1 head of the mushroom structure. The
F1 head contains the catalytic ability to make ATP. The F1
head is hexameric in structure with paired alpha and beta
Electron Transport
mitochondrion. The intact inner membrane of the mitochondrion

keeps the protons out, except for those that re-enter through
ATP Synthase. The ATP Synthase allows protons to re-enter the
mitochondrial matrix and harvests their energy to make ATP.
ATP Synthase
The ATP Synthase itself is an amazing nanomachine that makes
ATP using a gradient of protons flowing through it from the
intermembrane space back into the matrix. It is not easy to
depict in a single image what the synthase does. The figure at
the right illustrates the multi-subunit nature of this membrane
protein, which acts like a turbine at a hydroelectric dam. The
movement of protons through the ATP Synthase causes it to
spin like a turbine, and the spinning is necessary for making ATP.
ATP Synthase
34
proteins arranged in a trimer of
as a result of the proton
dimers. Movement of the gamma
excess in the intermembrane
protein inside the alpha-beta trimer
space, ATP is made.
causes each set of beta proteins to

Photosynthesis Lecture on
YouTube
change structure slightly into three
dierent forms called Loose, Tight,
The third type of phosphorylation to make ATP is found only in
and Open (L,T,O). Each of these
cells that carry out photosynthesis. This process is similar to
forms has a function. The Loose form
oxidative phosphorylation in several ways. A primary dierence is
binds ADP + Pi. The tight form
the ultimate source of the energy for ATP synthesis. In oxidative
squeezes them together to form the
phosphorylation, the energy comes from electrons produced by
ATP. The open form releases the ATP
oxidation of biological molecules. In the case of photosynthesis,
into the mitochondrial matrix. Thus,
the energy comes from the light of the sun.
Three States of
ATP Synthase
Electron Movement in Photophosphorylation
35
Energy Considerations in Photophosphorylation

from Wikipedia
Photons from the sun interact with chlorophyll molecules in

reaction centers in the chloroplasts of plants or membranes of
photosynthetic bacteria. A schematic of the process is shown
above.
The similarities of photophosphorylation to oxidative
phosphorylation include:
an electron transport chain
creation of a proton gradient
harvesting energy of the proton gradient by making ATP
with the help of an ATP synthase.
the source of the electrons H2O for photosynthesis versus

NADH/FADH2 for oxidative phosphorylation
direction of proton pumping into the thylakoid space of
the chloroplasts versus outside the matrix of the
mitochondrion
movement of protons during ATP synthesis out of the
thylakoid space in photosynthesis versus into the
mitochondrial matrix
nature of the terminal electron acceptor NADP+ in
photosynthesis versus O2 in oxidative phosphorylation.
Some of the dierences include :
36
Electron Transport in Chloroplasts versus

Mitochondria
In some ways, the movement of electrons in chloroplasts during
photosynthesis is opposite that of electron transport in
mitochondria. In photosynthesis, water is the source of electrons
and their final destination is NADPH. In mitochondria, NADH/
FADH2 are electron
Photosynthesis left me aghast
As electrons all went whizzing past
Leaving water at start
They trace the Z chart
In the membranes of each chloroplast
sources and H2O is

their final destination.
How do biological
systems get electrons
to go both ways? It
would seem to be the
equivalent of going to and from a particular place while always

going downhill, since electrons will move according to potential.
The answer is the captured energy of the photons, which elevates
electrons in photosynthesis to an energy where they move
downhill to their NADPH destination in a Z-shaped scheme
(previous page). The movement of electrons through this scheme
in plants requires energy from photons in two places to lift the
energy of the electrons suciently.
Last, it should be noted that photosynthesis actually has two
phases, referred to as the light cycle (described above) and the
dark cycle, which is a set of chemical reactions that captures CO2
from the atmosphere and fixes it, ultimately into glucose. The
Glycolysis and Gluconeogenesis

37
Energy Eciency
N-A-D
Cells are not 100% ecient in energy
To the tune of Penny Lane
use. Nothing that we know of is.

In the catabolic pathways that our cells employ
Oxidations help create the ATP
While they lower Gibbs free energy
Thanks to enthalpy
And the latter is a problem anaerobically

Cuz accumulations of it muscles hate
They respond by using pyruvate
To produce lactate
If a substrate is converted from an alcohol

To an aldehyde or ketone it is clear
Those electrons do not disappear
They just rearrange very strange
Catalyzing is essential for the cells to live

So the enzymes grab their substrates eagerly
If they bind with high affinity
Low Km you see, just as me
N-A-D is in my ears and in my eyes

Help-ing mol-e-cules get oxidized
Making N-A-D-H then
N-A-D is in my ears and in my eyes

Help-ing mol-e-cules get oxidized
Making N-A-D-H then
Consequently, cells do not get as

much energy out of catabolic
processes as they put into anabolic
processes. A good example is the
synthesis and breakdown of glucose,
something liver cells are frequently
doing. The complete conversion of
glucose to pyruvate in glycolysis
(catabolism) yields two pyruvates
plus 2 NADH plus 2 ATPs.
Conversely, the complete conversion
of two pyruvates into glucose by
gluconeogenesis (anabolism)
requires 4 ATPs, 2 NADH, and 2

GTPs. Since the energy of GTP is
essentially equal to that of ATP,
Recorded by Tim Karplus
dark cycle is also referred to as the Calvin Cycle and is discussed

HERE.
gluconeogenesis requires a net of 4

ATPs more than glycolysis yields. This
dierence must be made up in order
for the organism to balance everything. It is for this reason that

we eat. In addition, the ineciency of our capture of energy in
reactions results in the production of heat and helps to keep us
38
warm. You can read more about glycolysis and

gluconeogenesis HERE.
The Muscle Energy Song

To the tune of I Will
Metabolic Controls of Energy

It is also noteworthy that cells do not usually have
both catabolic and anabolic processes for the same
molecules (for example, breakdown of glucose and
synthesis of glucose, shown on the previous page)
occurring simultaneously inside of them because the
cell would see no net production of anything but heat
and a loss of ATPs with each turn of the cycle. Such
cycles are called futile cycles and cells have controls
in place to limit the extent to which they occur. Since
For running and for jumping

You need some energy
Chemically the body stores it
In the form of ATP
Ready whenever you are ever

Wanting to exercise
Steady as ever when whatever
Energy needs arise
If backup should be needed

Reserves are there in wait
Muscles brimming with supplies of
Tiny creatine phosphate
The action is exacting

For leaping in the air
Myofibrils all contracted
Using energy extracted
futile cycles can, in fact, yield heat, they are sources of
From reactions that react in me

Using A-T-P
You see
heat in some types of tissue. See also HERE for more

on futile cycles.
Molecular Backups for Muscles

For plants, the needs for energy are dierent than for
animals. Plants do not need to access energy

sources as rapidly as animals do, nor do they have to
maintain a constant internal temperature. Plants can
neither flee predators, nor chase prey. These needs of
animals are much more immediate and require that
energy stores be accessible on demand. Muscles, of
course, enable the motion of animals and the energy

39
required for muscle contraction is ATP. To have stores of energy
level, this means fighting a continual battle with entropy, but it is
readily available, muscles have, in addition to ATP, creatine
not the only need for energy that cells have.
phosphate and glycogen for quick release of glucose from

glycogen. The synthesis of creatine phosphate is a prime
example of the eects of concentration on the synthesis of high
energy molecules. For example, creatine phosphate has an
energy of hydrolysis of -43.1 kJ/mol whereas ATP has an energy
of hydrolysis of -30.5 kJ/mol Creatine phosphate, however, is
made from creatine and ATP in the reaction shown below. How is
this possible?
Creatine + ATP <=> Creatine phosphate + ADP
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
The G of this reaction is +12.6 kJ/mol, reflecting the energies

noted above. In a resting muscle cell, ATP is abundant and ADP
is low, driving the reaction to the right, creating creatine
phosphate. When muscular contraction commences, ATP levels
fall and ADP levels climb. The above reaction then reverses and
proceeds to synthesize ATP immediately. Thus creatine
phosphate acts like a battery, storing energy when ATP levels are
high and releasing it almost instantaneously to create ATP when
its levels fall.
Summary
In summary, energy is needed for cells to perform the functions
that they must carry out in order to stay alive. At its most basic
40
Chapter 3
Structure &
Function
Function flows from structure. In order to
understand the function of biomolecules, we must
first understand their structures.
Chapter 3
Structure and Function

Building Blocks
Proteins

Primary Structure

Secondary Structure

Alpha helix

Beta Strands

Fibrous Proteins

Ramachandran Plots

Tertiary Structure

Hydrophobic Eects

Quaternary Structure

Other Protein Structural Features

Cooperativity

Bohr Eect

2,3 BPG

Denaturation

Forces Stabilizing Structure

Refolding Denatured Proteins

Irreversible Denaturation

Prions and Misfolding
Nucleic Acids

Superhelicity

RNA Structures

Denaturing Nucleic Acids
Carbohydrates

Monosaccharides

Stereoisomer Nomeclature

Boat/Chair Conformations

Oligosaccharides

Polysaccharides

Amylose/Amylopectin

Glycogen

Cellulose

Glycosaminoglycans
Lipids and Membranes

Fatty Acids

Membrane Lipids

Lipid Bilayers

Membrane Proteins

Membrane Transport

Sodium-Potassium ATPase

Bacteriorhodopsin

Fat Soluble Vitamins

If we hope to understand function in biological systems, we must first understand

structure. At a simple level, we can divide molecules up according to their
anities for water hydrophobic (limited solubility in water), hydrophilic (soluble
in water) and amphiphilic (have characteristics of both hydrophobicity and
hydrophilicity). Hydrophobicity in biological molecules arises largely because
carbon-hydrogen bonds have electrons that are fairly evenly shared (not unlike
carbon-carbon bonds). By contrast, the electrons between the oxygen and
hydrogen of water are not equally shared. Oxygen has a greater electronegativity,
so it holds them closer than hydrogen does. As a consequence, oxygen has what
we call a partial negative charge and hydrogen has a partial positive charge.
Virtually all of life on Earth is built upon the biochemistry that arises from the
molecular properties described in the preceding paragraph. The biomolecules
referred to as lipids are largely water insoluble because they have predominantly
carbon-hydrogen bonds with few ionic or hydrogen bond characteristics.
Building Blocks
Biological macromolecules are all polymers of a
sort, even fats, in which the fatty acids can be
thought of as polymers of carbon. (We will
consider fatty compounds - fats,

YouTube lecture on amino
acids
42
glycerophospholipids, sphingolipids, isoprenoids/
The Amino Alphabet Song
terpenoids separately).
To the tune of Twinkle, Twinkle Little Star
The remaining categories of biological macromolecules include

proteins, nucleic acids, and polysaccharides. The building
Lysine, arginine and his

Basic ones you should not miss
Ala, leu, val, ile, and met
Fill the aliphatic set
Proline bends and cys has s
Glycines R is the smallest
Then theres trp and tyr and phe
Structured aromatically
blocks of these, respectively, are amino acids, nucleotides,

and monosaccharides (sugars). Of these, the most diverse
collection of chemical properties is found among the amino
acids.
Proteins
Whereas nucleotides all are water soluble and have the same
basic composition (sugar, base, phosphate) and the sugars
Asp and glus side chains of R

Say to protons au revoir
Glutamine, asparagine
Bear carboxamide amines
Threonine and tiny ser
Have hydroxyl groups to share
These twen-TY amino As
Can combine a zillion ways
also are water soluble and mostly contain 5 or 6 carbons (a few

exceptions), the amino acids (general structure below) are
structurally and
chemically diverse.
Though all of the amino
acids are, in fact, soluble
in water, the interactions
of their side chains with
water dier significantly.

This is important,
because it is only in the
side chains (R-groups)
that amino acids dier
from each other. Based
Amino Acid Schematic

from Wikipedia
43
on side chains, we can group the 20 amino acids found in
one need only consider the
proteins as follows:
positioning of the R-groups
Aromatic (phenylalanine, tyrosine, tryptophan)

Aliphatic (leucine, isoleucine, alanine, methionine, valine)
Hydroxyl/Sulfhydryl (threonine, serine, tyrosine, cysteine)
Carboxyamide (glutamine, asparagine)
R-Acids (glutamic acid, aspartic acid)
R-Amines (lysine, histidine, arginine)
Odd (glycine, proline)
around each peptide bond when

determining protein structure
Click HERE and HERE, for

Kevins Protein Structure
lectures on YouTube
schematically. Proteins that are

in aqueous environments, such as the cytoplasm of the cell, have
their amino acids arranged so that those with hydrophilic side
chains (such as threonine or lysine) predominate on the exterior
of the protein so as to interact with water. The hydrophobic
amino acids in these proteins are found predominantly on the
Note that tyrosine has a hydroxyl group and fits into two
categories. Note also that biochemistry books vary in how they
interior. When one examines the structure of proteins in nonaqueous environments, such as the interior of a lipid bilayer, the
organize amino acids into categories.
arrangement is flipped hydrophobics predominate on the
Amino acids are joined to each other by peptide bonds. This
of membrane fatty acids and the hydrophilic amino acids are
introduces a slight simplifying aspect to the structure of proteins
arranged anyplace where they can contact water. For a protein
outside where they can interact with the hydrophobic side chains
like porin, which provides an interior channel

through which water can pass, this is where the
hydrophilics are found. For transmembrane
proteins, which project through both sides of the
membrane, the hydrophilics are found at each point
where the polypeptide chain emerges from the
membrane.
Primary Structure
How do proteins obtain such arrangements of

44
amino acids? As we shall see, the
within about ten units of each other give
structures of all proteins ultimately
rise to regular repeating structures. These
arise from their amino acid
secondary structures include the well
sequences. The amino acid
known alpha-helix and beta strands. Both
sequence is referred to as the
were predicted by Linus Pauling, Robert
primary structure and changes in it
Corey, and Herman Branson in 1951.
can aect every other level of
Each structure has unique features. We use
structure as well as the properties of
the terms rise, repeat, and pitch to
a protein. The primary structure of a
describe the parameters of a helix. The
protein arrived at its current state as a
repeat is the number of residues in a helix
result of mutation and selection over
before it begins to repeat itself. The rise is
evolutionary time. On a more
the distance the helix elevates with addition
immediate time scale, 3D protein
of each residue. The pitch is the distance
structure arises as a result of a
between the turns of the helix.
phenomenon called folding. Protein

folding results from three dierent
Alpha Helix
structural elements beyond primary
The alpha helix (left) forms as a result of
structure. They are referred to as
interactions between amino acids
secondary, tertiary, and quaternary
separated by four residues. Interestingly,
structures, each arising from
the side chains of the amino acids in an
interactions between progressively
alpha helix are all pointed outwards from
more distant amino acids in the
the axis of the helix. Alpha helices have a
primary structure.
repeat of 3.6 amino acid residues per turn

of the helix, meaning that four turns of the
Secondary Structure
Interactions between amino acids
helix have approximately 14 amino acid

from Wikipedia
residues. Hydrogen bonds occur between

45
sheets. Other regular structures are also

known. What determines whether a given
stretch of a protein is in a helical or other
structure? Here is where the shape and
chemistry of the side chains plays a role.
Fibrous Proteins
Not all proteins have significant amounts of
tertiary or quaternary structure. (As we shall
see, these last two levels of structure arise
from bends in the polypeptide chains and
A Beta Sheet
from Wikipedia
the C=O of one amino acid and the N-H of another amino acid
interactions between separate polypeptide

chains, respectively.)
four residues distant and these help to stabilize the structure
Alpha keratin, for example, is what we refer
(note that the C=O and N-H involved are part of the polypeptide
to as a fibrous protein (also called
backbone, not the R-groups) Some amino acids have high helix
scleroprotein). Alpha keratin has primary
forming tendencies. They include methionine, alanine, leucine,
structure and secondary structure, but little
uncharged glutamate, and lysine. Others, such as proline,
tertiary or quaternary structure.
glycine, and negatively charged aspartate, disfavor its formation.
Consequently, alpha keratin exists mostly as

long fibers, such as are
Beta Strands
Beta strands are the most fundamental
helix, having essentially a 2D backbone of
folds like those of the pleats of a curtain.
Indeed, beta strands can be arranged
together to form what are called beta
We wouldnt be too popular

If keratins were globular
Our nails and hair would be as knots
Their structures folded up like clots
No strength theyd have and oh by gosh
Theyd rearrange with every wash
found in hair. Betakeratin is a harder

fibrous protein found in
nails, scales, and claws.
It is made up mostly of
Collagen
from Wikipedia
46
beta sheets. Proline, which is the least flexible
The carbonyl oxygen of the peptide bond can exist in
amino acid, due to attachment of the side chain
Kevins Protein Structure
resonance with the C-N bond, giving the peptide bond
to the alpha-amino group, is less likely to be
Lectures on YouTube
found in alpha helices, but curiously it is found
characteristics of a double bond and imposing

limitations for rotation around it. If we treat the
abundantly in the fibrous protein known as
peptide bond as a double bond, then the arrangements
collagen. Collagen (previous page) is the most abundant protein
of adjacent carbon bonds around it can be thought of as being in
in the human body and is the glue that literally sticks us
the cis or trans configurations. In proteins, not surprisingly, the
together. How does the inflexibility of proline permit it to be in a
preferred arrangement of these groups is strongly trans (1000/1).
helix? The answer is probably the parallel abundance in collagen
Of the 20 amino acids, the one that favors peptide bonds in the
of glycine, which contains the smallest side group and therefore
cis configuration most commonly is proline, but even for proline,
has the greatest flexibility.
the trans isomer is strongly preferred.
An interesting sidelight of the presence of proline in collagen is
Ramachandran Plots
the chemical modification of prolines, by the addition of hydroxyl

groups, after the protein is made. Such post-translational
Another consequence of considering the peptide bond as a

double bond is that it reduces the number of variable rotational
modifications are not uncommon. Threonine, serine, and

tyrosine frequently have their hydroxyl side-chains
phosphorylated. Lysines in collagen too are hydroxylated
post-translationally. The hydroxylated prolines and lysines
play a role in the formation of interchain hydrogen bonds
and crosslinking of triple helices during the assembly of
collagen fibrils. These bonds provide structural integrity to
the collagen. The enzymes that add hydroxyls to proline
and lysine require vitamin C (ascorbic acid) for their
activity. Lack of vitamin C leads to the production of
Peptide Bond Resonance and Structure
weakened collagen fibrils, resulting in a condition called

scurvy.
47
angles of the polypeptide backbone.

The terms phi and psi refer to
rotational angles about the bonds
between the N-alpha carbon and
Tertiary Structure
In contrast to secondary structures, which arise from interactions
between amino acids close in primary structure, tertiary
structure arises from interactions between amino
alpha carbon-
acids more distant in primary structure. Such
carbonyl
interactions are not possible in an endlessly
carbon
stretching fiber because each amino acid placed
respectively
between two amino acids causes them to be
(previous page).
moved farther away from each other in what is
Given the
essentially the two dimensions of a secondary
bulkiness of R-
structure. For distant amino acids to interact, they
groups, the
must be brought into closer proximity and this
phenomenon of
Phi and Psi Angles
from Wikipedia
requires
steric
bending
hindrance and
the tendency of
Ramachandran Plot
close side
chains to interact with each other, one might expect to find a bias
in the values of phi and psi. Indeed, that is exactly what is
observed. Dr. G.N. Ramachandran proposed such a result and,
in a plot that bears his name, depicted the theoretical likelihood of
each angle appearing in a polypeptide. More recent observations
of actual phi and psi angles in data from the PDB protein
database bear out Dr. Ramachandrans predictions. In the plot
above, beta strands fit nicely in the green section at the top and
alpha helices fit in the green section in the middle.
and
folding of
the
polypeptide chain.
Proteins with such
structures are referred to
as globular and they
are, by far, the most
abundant class of
proteins. Indeed, it is in
globular proteins that
we have the most vivid
The Structure of Myoglobin

from Wikipedia
48
images of the results of folding.
O Little Protein Molecule
Folds in polypeptides arise as a result
To the tune of O Little Town of Bethlehem
of bends between regions of secondary
Oh little protein molecule

You're lovely and serene
With twenty zwitterions like
Cysteine and alanine
A folded enzymes active

And starts to catalyze
When activators bind into
The allosteric sites
Your secondary structure

Has pitches and repeats
Arranged in alpha helices
And beta pleated sheets
Some other mechanisms

Control the enzyme rates
By regulating synthesis
And placement of phosphates
The Ramachandran plots are

Predictions made to try
To tell the structures you can have
For angles phi and psi
And tertiary structure

Gives polypeptides zing
Because of magic that occurs
In protein fol-ding
And all the regulation

That's found inside of cells
Reminds the students learning it
Of pathways straight from hell
structure (such as alpha helix or beta

strands). Such structures may be
preferred due to incompatibility of a
given amino acid side chain for a
secondary structure formed by the amino
acids preceding it. Bends occur
commonly in proteins and proline is often
implicated.
Bends do not have the predictable
geometry of alpha helices or beta
strands and are often referred to as
random coils. Thus, even though protein
structure can be described easily as
regions of secondary structure separated
by bends, the variability of bend
structures makes prediction of tertiary
structure from amino acid sequence

enormously more dicult than
identifying/predicting regions of
secondary structure.
49
To understand the hydrophobic eect, perform the following
Hydrophobic
Eect
experiment take the water-oil mixture and shake it vigorously.

This will force the layers to mix and one will observe that tiny
It is at the level of tertiary
globules of both water and oil can, in fact, be found initially in the
structure that the
layer of each. Over time, though, the tiny globules break up and
characteristic arrangement
merge with the appropriate layer. This is due to the phenomenon
of hydrophobic and
of entropy and consideration of surface area. First, the sum of
hydrophilic amino acids in a

protein occurs. In an
aqueous environment, for a
protein to remain soluble, it
must have favorable
the surface area of the embedded tiny globs is far greater than
A hydrophobic effect causes the
water on this leaf to assume a
the area of the region between the two layers after mixing is over.
The smaller the globs, the more the surface area of interaction
spherical shape
from Wikipedia
interactions with the water
Interactive 3.1
around it, hence, the positioning of hydrophilic amino acids

externally. Another impetus for the folding phenomenon is a bit
harder to understand. It is known as the hydrophobic eect. At
a chemical level, it makes sense hydrophobic amino acids will
prefer to interact with each other internally and away from water.
The driving force for this phenomenon, though, is a bit more
conceptually dicult. Consider a bottle containing oil and water.
As everyone knows, the two liquids will not mix and instead will
form separate layers. A reasonable question might be why they
do this instead of one existing as tiny globules inside of the other.
The answer to that question, as well as the positioning of
hydrophobic amino acids in the interior of water soluble proteins,
is the hydrophobic eect.
Oxygenated Myoglobin
50
hydrophobic layers and, as a consequence, more ordering. Since
Interactive 3.2
entropy in a closed system tends to increase, it will tend to

reduce the amount of ordering, if left alone. Thus, one can
increase the ordering on a nanoscopic scale (forming globules) by
applying energy in the form of shaking. When left alone, however,
the system will increase its disorder by reducing the interactions
between hydrophobic groups and hydrophilic ones.
In the oil water mixture, this causes the tiny globs to break up and
produce the two
layers we are
familiar with
because this is
the minimum
surface area
Hemoglobin in the Absence of Oxygen
that can be
made between
between the oil and the water. The minimum possible surface
the two layers
area of interaction occurs when there are no globs at all just
and thus the
two layers and nothing else.
least ordering.
How does this relate to entropy? Interactions between the
In proteins,
water-hydrophobic layers causes the molecules at the interface
hydrophobic
to arrange themselves precisely/regularly so as to minimize
amino acid side
their interactions. Ordering thus occurs at the layer interfaces.
chains are
The maximum amount of ordering occurs when the maximum
shielded from
surface areas of oil and water interact. Small globules give rise
water by
to more exposed surface area between the water and
placement
51
Quaternary
Structure
The last level of
protein structure we
will consider is that of
My blood has a proclivity

For co-op-ER-a-TIV-it-y
Its cause when in the lung environs
Ox-y-GEN binds to the irons
And changes hemoglobins fate
Out of a T to the R-State
quaternary structure.
In order to have quaternary structure, a protein must have
multiple polypeptide subunits because the structure involves the
arrangement of those subunits with respect to each other.
Consider hemoglobin, the oxygen-carrying protein of our blood.
It contains two identical subunits known as alpha and two other
identical ones known as beta. These are arranged together in a
fashion as shown on the previous page. By contrast, the related
O2 and CO Binding to Heme
internal to the protein, thus also reducing interfaces between

hydrophobic residues and water. In both cases, entropy is
increased, due to the reduced organization of the layers. Once
formed, the interactions between the hydrophobic amino acid
side chains helps to stabilize the overall protein structure.

Kevins Hemoglobin lectures
on YouTube
Heme and Protoporphyrin Ring

52
oxygen storage protein known as myoglobin only contains a
that binding of one ligand molecule by a protein favors the
single subunit. Hemoglobin has quaternary structure, but
binding of additional molecules of the same type. Hemoglobin, for
myoglobin does not. Multiple subunit proteins are common in
example, exhibits cooperativity when the binding of an oxygen
cells and they give rise to very useful properties not found in
molecule by the iron of the heme group in one of the four
single subunit proteins. In the case of hemoglobin, the multiple
subunits causes a slight conformation change in the subunit.
subunits confer the property of cooperativity variable anity for
This happens because the heme iron is attached to a histidine
oxygen depending on the latters concentration. In the case of
side chain and binding of oxygen lifts the iron along with the
enzymes, it can impart allosterism the ability to have the
histidine ring (also known as the imidazole ring).
activity of the enzyme altered by interaction with an eector

molecule. We will discuss allosterism in detail in the next chapter.
Other Protein Structural Features

Not everything found in a protein is an amino acid. Proteins
frequently have other chemical groups, known as prosthetic
groups, bound to them, that are necessary for the function of a
protein. Examples include the porphyrin ring (below) of heme in
myoglobin and hemoglobin that carries an iron so that oxygen
can be bound. Metals are frequently employed by enzymes in
their catalysis. Several vitamins (referred to as coenzymes),
Since each hemoglobin subunit interacts with and influences the

other subunits, they too are induced to change shape slightly
when the first subunit binds to oxygen (a transition described as
going from the T-state to the R-state). These shape changes favor
each of the remaining subunits binding oxygen, as well. This is
very important in the lungs where oxygen is picked up by
hemoglobin, because the binding of the first oxygen molecule
facilitates the rapid uptake of more oxygen molecules. In the
tissues, where the oxygen concentration is lower, the oxygen
leaves hemoglobin and the proteins flips from the R-state back to
the T-state.
such as thiamine (B1) and riboflavin (B2) are modified and

chemically bound to enzymes to help them perform specific
Cooperativity is only one of many fascinating structural aspects of
catalytic functions.
hemoglobin that help the body to receive oxygen where it is

needed and pick it up where it is abundant. Hemoglobin also
Cooperativity
An interesting and important aspect of some proteins is the
phenomenon of cooperativity. Cooperativity refers to the fact
assists in the transport of the product of cellular respiration

(carbon dioxide) from the tissues producing it to the lungs where
it is exhaled. Let us consider these individually.
53
require oxygen and release protons and carbon dioxide. The

higher the concentration of protons and carbon dioxide, the
more oxygen is released to feed the tissues that need it most.
2,3 BPG
Another molecule aecting the release of oxygen by
hemoglobin is 2,3 bisphosphoglycerate (also called 2,3 BPG or
just BPG). Like protons and carbon dioxide, 2,3 BPG is
produced by actively respiring tissues, as a byproduct of
glucose metabolism. The 2,3
BPG molecule fits into the
hole of the donut of adult
hemoglobin. Such binding of
2,3 BPG favors the T (tight)
The Bohr Effect
state of hemoglobin, which

has a reduced anity for
oxygen. In the absence of
Bohr Eect
2,3 BPG, hemoglobin can exist
The Bohr Eect was first described over 100 years ago by
in the R (relaxed) state, which
2,3 BPG
Christian Bohr. Shown graphically (above left), the observed
has a high anity for oxygen.
eect is that hemoglobins anity for oxygen decreases as the pH

decreases and/or as the concentration of carbon dioxide
Fetal Hemoglobin
increases. Binding of the protons by histidine helps to facilitate
Adult hemoglobin releases oxygen when it binds 2,3 BPG. This is
structural changes in the protein and also with the uptake of
in contrast to fetal hemoglobin, which has a slightly dierent
carbon dioxide. Physiologically, this has great significance
configuration (22) than adult hemoglobin (22). Fetal
because actively respiring tissues (such as contracting muscles)
hemoglobin has a greater anity for oxygen than maternal

54
Hemoglobins Moving Around

To the tune of Santa Claus is Coming to Town
Oh isn't it great
What proteins can do
Especially ones that bind to O2
Hemoglobin's moving around
The proton concentration

Is high and has a role
Inside of the lungs

It picks up the bait
To empty their loads

The globins decree
"We need to bind 2,3BPG"
Between the alpha betas

It finds imidazole
And changes itself from T to R state

hemoglobin,
allowing the
fetus to obtain
oxygen
eectively from
the mothers
blood. Part of
the reason for
fetal
hemoglobins
greater anity
Arising when an O2 binds

Pulling up on histidine
The stage is thus set

For grabbing a few
Cellular dumps of CO2
The binding occurs

Cooperatively
And then inside the lungs it

Discovers ox-y-gen
bind 2,3 BPG.
Thanks to changes qua-ter-nar-y

And dumps the CO2 off

To start all o'er agin
It exits the lungs

Engorged with O2
So see how this works

You better expect
In search of a working body tissue

To have to describe the Bohr effect

The proto-porphyrin system

Its iron makes such a scene
for oxygen is
that it doesnt
Fetal Hemoglobin Binding of O2
Another significant fact about 2,3 BPG is that its

concentration is higher in the blood of smokers than it is
of non-smokers. Consequently, hemoglobin in a smokers
blood spends more time in the T state than the R state.
That is a problem when it is in the lungs, where being in
the R state is necessary to maximally load the hemoglobin
with oxygen. A high blood level of 2,3 BPG is one of the

reasons smokers have trouble breathing when they
exercise they have reduced oxygen carrying capacity.
Last, though it is not related directly to 2,3 BPG, smokers

have another reason why their oxygen carrying capacity is
55
lower than that of non-smokers. Cigarette
Forces Stabilizing Structure
smoke contains carbon monoxide and this
Amino acids are linked one to the other by
molecule, which has almost identical
peptide bonds. These covalent bonds are
dimensions to molecular oxygen, competes
extraordinarily stable at neutral pHs, but can
eectively with oxygen for binding to the iron
be broken by hydrolysis with heat under
atom of heme. Part of carbon monoxides
acidic conditions. Peptide bonds, however,
toxicity is due to its ability to bind hemoglobin
only stabilize primary structure and, in fact,
and prevent oxygen from binding.
are the only relevant force responsible for it.

Secondary structure, on the other hand, is
Denaturation
generally stabilized by weaker forces,
For proteins, function is dependent on precise
including hydrogen bonds. Hydrogen bonds
structure. Loss of the precise, folded structure
are readily disrupted by heat, urea, or
of a protein is known as denaturation and is
guanidinium chloride.
usually accompanied by loss of function.

Anyone who has ever worked to purify an
Forces stabilizing tertiary structure include
enzyme knows how easy it is for one to lose its
ionic interactions, disulfide bonds,
activity. A few enzymes, such as ribonuclease,
hydrophobic interactions, metallic bonds, and
are remarkably stable under even very harsh
hydrogen bonds. Of these, the ionic
conditions. For most others, a small
interactions are most sensitive to pH
temperature or pH change can drastically aect
changes. Hydrophobic bonds are most
activity. The reasons for these dierences vary,
sensitive to detergents. Thus, washing ones
but relate to 1) the strength of the forces holding
hands helps to kill bacteria by denaturing
the structure together and 2) the ability of a
critical proteins they need to survive. Metallic
protein to refold itself after being denatured. Let
bonds are sensitive to oxidation/reduction.
us consider these separately below.
Breaking disulfide bonds requires either a
strong oxidizing agent, such as performic

56
acid or a strong reducing agent on another disulfide, such as

mercaptoethanol or dithiothreitol.
Quaternary structures are stabilized by the same forces as tertiary
structure and have the same sensitivities.
Refolding Denatured Proteins

All of the information for protein folding is contained in the
primary structure of the protein. It may seem curious then that
most proteins do not refold into their proper, fully active form after
they have been denatured and the denaturant is removed. A few
do, in fact, refold correctly under these circumstances. A good
example is bovine ribonuclease (also called RNase). Its catalytic
activity is very resistant to
There are not very many ways
Inactivating RNase
Its stable when its hot or cold
Because disulfides tightly hold
If you desire to make it stall
Use hot mercaptoethanol
heat and urea. However, if

one treats the enzyme with
mercaptoethanol (which
breaks disulfide bonds)
from Wikipedia
Prion Protein Misfolding
prior to urea treatment and
Irreversible Denaturation
heating, activity is lost,
Most enzymes, however, do not behave like ribonuclease. Once
indicating that the covalent
denatured, their activity cannot be recovered to any significant
disulfide bonds help stabilize the overall enzyme structure. If one
extent. This may seem to contradict the idea that folding
allows the enzyme mixture to cool back down to room
information is inherent to the sequence of amino acids in the
temperature, over time some enzyme activity reappears,
protein. It does not. The reason most enzymes cant refold
indicating that ribonuclease can re-fold under the proper
properly is due to two phenomena. First, normal folding may
conditions.
occur as proteins are being made. Interactions among amino

acids early in the synthesis are not confused by interactions
57
I think that if I chanced to be on

The protein making up a prion
Id twist it and for goodness sakes
Stop it from making fold mistakes
with amino acids

later in the synthesis
because those amino
acids arent present
as protein synthesis
starts. In many
cases, the proper folding of newly made polypeptides is also

assisted by special proteins called chaperones. Chaperones bind
to newly made proteins, preventing interactions that might result
in misfolding. Thus, early folding and the assistance of
chaperones eliminate some potential wrong-folding interactions
that can occur if the entire sequence was present when folding
started.
Denatured full-length polypeptides have many more potential
wrong folds that can occur. A second reason most proteins dont
refold properly after denaturation is probably that folding, like any
other natural phenomenon, is driven by energy minimization.
Though the folded structure may have a low energy, the path
leading to it may not be all downhill. Like a chemical reaction that
has energies of activation that must be overcome for the reaction
to occur, folding likely has peaks and valleys of energy that do not
automatically lead directly to the proper fold. Again, folding
during synthesis leads the protein along a better-defined path
through the energy maze of folding that denatured full-length
proteins cant navigate.
Prions and Misfolding

Folding and the stability of folded proteins is an important
consideration for so-called infectious proteins known as prions.
These mysterious proteins, which are implicated in diseases,
such as mad cow disease and the related human condition
known as Creutzfeldt-Jakob disease, result from the improper
folding of a brain protein known as PrP. The misfolded protein
has two important properties that lead to the disease. First, it
tends to aggregrate into large complexes called amyloid plaques
that damage/destroy nerve cells in the brain, leading ultimately to
dementia and loss of brain function.
Second, and probably worse, the misfolded protein induces
other copies of the same protein to misfold as well. Thus, a
misfolded protein acts something like a catalytic center and the
disease progresses rapidly. The question arises as to how the
PrP protein misfolds to begin with, but the answer to this is not
clear. There are suggestions that exposure in the diet to
misfolded proteins may be a factor, but this is disputed. An
outbreak of mad cow disease in Britain in the 1980s was followed
by a rise in the incidence of a rare form of human CreutzfeldJakob disease called variant CJD
(v-CJD), lending some credence
to the hypothesis. It is possible
that misfolding of many proteins
YouTube lecture on Nucleic
occurs sporadically without
Acid Structure
58
scientific impact per word than any other research article ever
published. Today, every high school biology student knows the
double helical structure in which G pairs with C and A pairs with
T. The DNA molecule is a polymer of nucleoside
Base Pairing Interactions
consequence or observation, but if PrP misfolds, the results are

readily apparent. Thus, Creutzfeld-Jakob disease may ultimately
give insights into the folding process itself.
Nucleic Acids
Determination of the structure of the most common form of DNA,
known as the B form, was one of the most important scientific
advances of the 20th century. Using data from Rosalind
Franklin, James Watson and Francis Crick initiated the modern
era of molecular biology with their paper in the April 25, 1953
issue of Nature. Arguably, that single page paper has had more
DNA Structure
59
monophosphates with phosphodiester bonds between the
Major Groovy
To the tune of Feelin Groovy
The DNA forms
A and B
Have bases
Complementary
Despite the similarities
They differ in their
Major groovies
Nanananananana major groovies
Transcription factors
With their bindin'
Cause DNA to
Start unwindin'
Holding it
Aggressively
By forming bonds
In major groovies
Nanananananana major groovies
For proteins, the key

To sequence I-D
Is hydrogen bonding, each base pair unique
Purine, pyrimidine patterns discrete
In DNA's most
Major groovy
Nanananananana major groovy
phosphate and the 5 end of one deoxyribose and the 3 end of

the next one. In the B form the DNA helix has a repeat of 10.5
base pairs per turn, with sugars and phosphate forming the
covalent backbone of the molecule and the adenine, guanine,
cytosine, and thymine bases oriented in the middle where they
form the now familiar base-pairs that look like the rungs of a
ladder.
Hydrogen bonds help to hold the base pairs together, with two
hydrogen bonds per A-T pair and three hydrogen bonds per GC pair. The two strands of a DNA duplex run in opposite

A, B, and Z Forms of DNA

from Wikipedia
60
resembles the B form,
Its taught in school of DNA

The race for structure underway
Gave rise to competition huge
Along with data subterfuge
In view of this we should extend
New authorship to make amends
A fairer order could be picked
Franklin, Wilkins, Watson, Crick
it turns out to be
important in the duplex
form of RNA and in
RNA-DNA hybrids.
Both the A form and
the B form of DNA
have the helix oriented
in what is termed the
right-handed form.
directions. The 5 end of one strand is

paired with the 3 end of the other strand
These stand in contrast
and vice-versa at the other end of the
to another form of
duplex. The B form of DNA has a
DNA, known as the Z
prominent major groove and a minor
form. Z-DNA, as it is
groove tracing the path of the helix (shown
known, has the same
at left). Proteins, such as transcription
base-pairing rules as
factors bind in these grooves and access
the B and A forms, but
the hydrogen bonds of the base pairs to
instead has the helices
read the sequence therein.
twisted in the opposite

direction, making a
Other forms of DNA besides the B form are
left-handed helix (see
known. One of these, the A form, was
figure on previous
identified by Rosalind Franklin in the same
page). The Z form has
issue of Nature as Watson and Cricks

paper. Though the A structure is a
relatively minor form of DNA and
a sort of zig-zag shape,
Topoisomers of DNA
from Wikipedia
giving rise to the name

61
B-DNA
To the tune of Y-M-C-A
Phosphates
Are in nucleotides
I say phosphates
Cover bases inside
I say phosphates
Span the 5 and 3 primes
Theres no need - to - be - real - mixed - up
Proteins
Full of amino As
I say proteins
Come from mRNAs
I say proteins
Require tRNAs
There is more you - need - to trans-late
Bases
Carry info you see
I say bases
Are all complementry
I say bases
Like A,T,G and C
They have got - to - be all - paired - up
Codons
Like our friend U-A-C
I say codons
Come in clusters of three
I say codons
Have one base wobble ee
Now you can - go - forth - and - tran-slate
Its fun to play with some B-DNA

Its got a boatload of G-C-T-A
Its got everything
A polymerase needs
When you melt all the As and Ts

With those hydrogen Bs
And right-hand he-li-ces
Anti-par-a-llel fives and threes

You can make RNAs
With a po-ly-mer-ase
Just by pairing up Us with As

With those hydrogen Bs
And right-hand he-li-ces
Anti-par-a-llel fives and threes
B-DNA

62
Z DNA. In addition,
the winding ahead of it. Unrelieved, such tension in a DNA
the helix is rather
duplex can result in structural obstacles to replication.
stretched out
compared to the A
and B forms. Why
are there dierent
forms of DNA?
The answer relates
to both
superhelical
3D tRNA Structure (left) and
2D projection (right)
tension and
sequence bias.
from Wikipedia
Sequence bias
means that certain
Such adjustments can occur in three ways. First, tension can

provide the energy for flipping DNA structure. Z-DNA can arise
as a means of relieving the tension. Second, DNA can supercoil
to relieve the tension. In this method, the strands of the duplex
can cross each other repeatedly, much like a rubber band will coil
up if one holds one section in place and twists another part of it.
Third, enzymes called topoisomerases can act to relieve or, in
some cases, increase the tension by adding or removing twists in
the DNA.
RNA Structures
sequences tend to favor the flipping of B form DNA into other
With respect to structure, RNAs are more varied than their DNA
forms. Z DNA forms are favored by long stretches of alternating
cousins. Created by copying regions of DNA, cellular RNAs are
Gs and Cs. Superhelical tension will be discussed below.
synthesized as single strands, but they often have selfcomplementary regions leading to fold-backs containing duplex
Superhelicity
regions. The structure of tRNAs and rRNAs are excellent
Short stretches of linear DNA duplexes exist in the B form and
examples. The base-pairing rules of DNA are the same in RNA
have 10.5 base pairs per turn. Double helices of DNA in the cell
can vary in the number of base pairs per turn they contain. There
are several reasons for this. For example, during DNA replication,
(with U in RNA replacing the T from DNA), but in addition, base

pairing between G and U can also occur in RNA. This latter fact
leads to many more possible duplex regions in RNA that can exist
strands of DNA at the site of replication get unwound at the rate
compared to single strands of DNA.
of 6000 rpm by an enzyme called helicase. The eect of such
RNA structure, like protein structure, has importance, in some
local unwinding at one place in a DNA has the eect increasing
cases, for catalytic function. Like random coils in proteins that

63
give rise to tertiary

structure, singlestranded regions of
RNA that link duplex
regions give these
molecules a tertiary
structure, as well.
Catalytic RNAs,
called ribozymes,
catalyze important
cellular reactions,
Hyperchromic Effect
including the
formation of peptide
bonds. DNA, which
is usually present in cells in strictly duplex forms (no tertiary

structure, per se), is not known to be involved in catalysis.
Denaturing Nucleic Acids

Like proteins, nucleic acids can be denatured. Forces holding
duplexes together include hydrogen bonds between the bases of
each strand that, like the hydrogen bonds in proteins, can be
broken with heat or urea. (Another important stabilizing force for
DNA arises from the stacking interactions between the bases in a
strand.) Single strands absorb light at 260 nm more strongly than
double strands (hyperchromic eect), allowing one to easily follow
denaturation. For DNA, strand separation and strand
hybridization are important aspects of the technique known as
the polymerase chain reaction (PCR). Strand separation of DNA
duplexes is accomplished in the method by heating them to
boiling. Hybridization is an important aspect of the method that
requires complementary single strands to find each other and
form a duplex. Thus, DNAs (and RNAs too) can renature readily,
unlike most proteins. Considerations for ecient hybridization
RNA structures are important for reasons other than catalysis.
(also called annealing) include temperature, salt concentration,
The 3D arrangement of tRNAs is important for enzymes that
strand concentration, and magnesium ion levels.
attach amino acids to them to do so properly. Small RNAs called

siRNAs found in the nucleus of cells appear to play roles in both
Carbohydrates
gene regulation and in cellular defenses against viruses. The key
The last class of macromolecules we will consider structurally
to the mechanisms of these actions is the formation of short fold-
here is the carbohydrates. Built of sugars or modified sugars,
back RNA structures that are recognized by cellular proteins and
carbohydrates have several important functions, including
then chopped into smaller units. One strand is copied and used
structural integrity, cellular identification, and energy storage.
to base pair with specific mRNAs to prevent the synthesis of

proteins from them.
64
Monosaccharides
Simple sugars, also known as monosaccharides, can generally be
written in the form Cx(H2O)x. It is for this reason they are referred
to as carbo-hydrates. By convention, the letters ose at the end
of a biochemical name flags a molecule as a sugar. Thus, there
are glucose, galactose, sucrose, and many other -oses. Other
descriptive nomenclature involves use of a prefix that tells how
many carbons the sugar contains. For example, glucose, which
contains six carbons, is described as a hexose. The following list
shows the prefixes for numbers of carbons in a sugar:
Tri- = 3
Ribose = aldo-pentose
Glucose = aldo-hexose
Galactose = aldo-hexose
Mannose = aldo-hexose
Glyceraldehyde = aldo-triose
Erythrose aldo-tetrose
Fructose = keto-hexose
Ribulose = keto-pentose
Sedoheptulose = keto-heptose
Dihydroxyacetone = keto-triose
Stereoisomer Nomenclature
Sugars of a given category (hexoses, for example) dier from
Tetr- = 4
each other in the stereoisomeric configuration of their carbons.
Pent- = 5
Two sugars having the same number of carbons (hexoses, for
Hex- =6
example) and the same chemical form (aldoses, for example), but
Hept- = 7
diering in the stereoisomeric configuration of their carbons are
Oct- = 8
Other prefixes identify whether the sugar contains an aldehyde

group (aldo-) or a ketone (keto) group. Prefixes may be
called diastereomers. Biochemists use D and L nomenclature to

describe sugars, as explained below.
combined. Glucose, which contains an aldehyde group, can be
D-sugars
described as an aldo-hexose. The list that follows gives some
predominate in
common sugars and some descriptors.
nature, though L-

Kevins YouTube lectures on
Carbohydrate Structure
forms of some
sugars, such as
fucose, do exist.
The D and L
designation is a
Diastereomers
65
bit more
complicated than
it would appear on
the surface. To
determine if a
sugar is a D-sugar
or an L-sugar, one
simply examines
the configuration
of the highest
Cyclization of Glucose
numbered
asymmetric
carbon. If the
hydroxyl is written
Ketose and Aldose (top)
Enantiomers (bottom)
are enantiomers, but D-Erythrose and D-Threose are

diastereomers.
to the right, it is a
Sugars of 5-7 carbons can fairly easily form ring structures
D-sugar. If the
(called Haworth structures). For aldoses like glucose, this
hydroxyl is on the
involves formation of a hemi-acetal. For ketoses like fructose, it
left, it is an L-
involves formation of a hemi-ketal. The bottom line for both is
sugar. That part is simple. The confusion about D and L arises
that the oxygen that was part of the aldehyde or the ketone
because L sugars of a given name (glucose, for example) are
group is the one that becomes a part of the ring. More important
mirror images of D sugars of the same name. The figure on the
than the oxygen, though, is the fact that the carbon attached to
previous page shows the structure of D- and L-glucose. Notice
it (carbon #1 in aldoses or #2 in ketoses) becomes asymmetric
that D-glucose is not converted into L-glucose simply by flipping
as a byproduct of the cyclization. This new asymmetric carbon
the configuration of the fifth carbon in the molecule. There is
is called the anomeric carbon and it has two possible
another name for sugars that are mirror images of each other.
configurations, called alpha and beta, as shown in the figure on
They are called enantiomers. Thus, L-glucose and D-glucose
the previous page.

66
Hark the Sucrose

To the tune of Hark the Herald
Carbohydrates all should sing

Glory to the Haworth ring
Anomeric carbons hide
When they're in a glycoside
Glycoside Synthesis
A solution of glucose will contain a mixture of alpha and beta

forms. Whether the alpha or the beta arises upon cyclization is
partly determined by geometry and partly random. Thus, one can
find a bias for one form, but usually not that form exclusively. A
Glucopyranose is there
In the boat or in the chair
Alpha, beta, D and L
Di-astere-omer hell
Alpha, beta, D and L
Di-astere-omer hell
given molecule of sugar will flip between alpha and beta over
time. A requirement for this is that the hydroxyl on the anomeric
carbon is unaltered, thus facilitating flipping back to the straight
chain form followed by recyclization. If the hydroxyl becomes
chemically altered in any way (for example, replacement of its

hydrogen by a methyl group), a glycoside is formed. Glycosides
are locked in the same alpha or beta configuration they were in
when the modification was made. Glycosides are commonly

found in nature. Sucrose, for example , is a di-glycoside both
The last considerations for sugars relative to their structure are
the glucose and the fructose have had their anomeric hydroxyls
their chemical reactivity and modification. The aldehyde group of
altered by being joined together.
aldoses is susceptible to oxidation, whereas ketoses are less so.

Sugars that are readily oxidized are called reducing sugars
because their oxidation causes other reacting molecules to be
67
reduced. Reducing sugars can easily be identified

in a chemical test. Chemical modification of sugars
occurs readily in cells. As we will see,
phosphorylation of sugars occurs routinely during

Carbohydrate Structure
Sugars are readily joined together (and broken

apart) in cells. Sucrose (right), which is common
table sugar, is made by joining the anomeric
metabolism. Oxidation of sugars to create carboxyl

groups also can occur. Reduction of aldehyde/ketone groups of
sugars creates what are called sugar
alcohols, and other modifications,
Disaccharides
hydroxyl of alpha-D-glucose to the anomeric hydroxyl of beta-Dfructose. Not all disaccharides join the anomeric hydroxyls of
both sugars. For example, lactose (milk sugar) is made by linking
such as addition of sulfates and
the anomeric hydroxyl of galactose in the beta configuration to
amines also readily occur.
the hydroxyl of carbon #4 of glucose.
Boat/Chair
Conformations
Oligosaccharides
Independent of stereoisomerization,
sugars of 5-15 units, typically. Oligosaccharides are not
sugars in ring form of a given type
commonly found free in cells, but instead are found covalently
The term oligosaccharide is used to describe polymers of
(such as glucose) can twist themselves into
attached to proteins, which are then said to be
alternative conformations called boat and chair.
glycosylated. Oligosaccharides attached to proteins
Note that this rearrangement does not change the
may be N-linked (through asparagine) or O-linked
relative positions of hydroxyl groups. All that has
(though serine or
changed is the shape of the molecule. As shown
threonine). O-
for glucose, one can see that the beta-hydroxyl of
linked sugars
glucose is closer to the CH2OH (carbon #6) in the
are added only in
boat form than it is in the chair form. Steric
the Golgi
hindrance can be a factor in favoring one
apparatus while
configuration over another.
N-linked sugars
Sucrose
are attached
An oligosaccharide
from Wikipedia
68
starting in the endoplasmic reticulum and then completed in the
lubrication. Polysaccharides involved in energy storage include
Golgi.
the plant polysaccharides, amylose and amylopectin. The
Oligosaccharides often function as identity markers, both of cells

and proteins. On the cell surface, glycoproteins with distinctive
oligosaccharides attached establish the identity of each cell. The
types of oligosaccharides found on the surface of blood cells is a
determinant of blood type.
The oligosaccharides that are attached to proteins may also
polysaccharide involved in energy storage in animals is called

glycogen and it is mostly
found in the muscles and
liver.
Amylose/
Amylopectin
Amylose
determine their cellular destinations. Improper glycosylation or
Amylose is the simplest
sugar modification patterns can result in the failure of proteins to
of the polysaccharides,
reach the correct cellular compartment. For example, inclusion
being comprised solely of glucose units joined in an alpha 1-4
cell (I-cell) disease arises from a defective phosphotransferase in
linkage. Amylose is broken down by the enzyme alpha-amylase,
the Golgi. This enzyme normally catalyzes the addition of a
found in saliva. Amylopectin is related to amylose in being
phosphate to a mannose sugar attached to a protein destined for
composed only of glucose, but it diers in how the glucose units
the lysosome. In the absence of a functioning enzyme, the
are joined together. Alpha 1-4 linkages predominate, but every
unphosphorylated glycoprotein never makes it to the lysosome
30-50 residues, a branch arises from an alpha 1-6 linkage.
and is instead exported out of the cell where it accumulates in the
Branches make the structure of amylopectin more complex than
blood and is excreted in the urine. Individuals with I-cell disease
that of amylose.
suer developmental delays, abnormal skeletal development, and

restricted joint movement.
Polysaccharides
Glycogen
Glycogen is a polysaccharide that is physically related to
amylopectin in being built only of glucose and in having a mix of
Polysaccharides, as their name implies, are made by joining
alpha 1-4 and alpha 1-6 bonds. Glycogen, however, has many
together many sugars. The functions for polysaccharides are
more alpha 1-6 branches than amylopectin, with such bonds
varied. They include energy storage, structural strength, and
occurring about every 10 residues. One might wonder why such

69
Cellulose
Another
important
polysaccharide
containing only
glucose is
cellulose. It is
Repeating structure of cellulose
a polymer of
from Wikipedia
glucose used to
give plant cell walls structural integrity and has the individual units
Glycogen Structure
joined solely in a beta 1-4 configuration. That simple structural

from Wikipedia
branching occurs more abundantly in animals than in plants. A

plausible explanation is based on the method by which these
molecules are broken down. The breakdown of these
polysaccharides is catalyzed by enzymes, known as
phosphorylases, that clip glucose residues from the ends of
change makes a radical dierence in its digestibility. Humans are

unable to break down cellulose and it passes through the
digestive system as roughage. Ruminant animals, such as cattle,
however have bacteria in their rumens that contain the enzyme
cellulase. It breaks the beta 1-4 links of the glucoses in cellulose
to release the sugars for energy.
glycogen chains and attach a phosphate to them in the process,
Another polysaccharide
producing glucose-1-phosphate. More highly branched
used for structural integrity
polysaccharides have more ends to clip, and this translates to
is known as chitin. Chitin
more glucose-1-phosphates that can be removed simultaneously
makes up the exoskeleton of
by numerous phosphorylases. Since glucose is used for energy
insects and is a polymer of a
by muscles, glucose concentrations can be increased faster the
modified form of glucose
more branched the glycogen is. Plants, which are immobile do
known as N-acetyl-
not have needs for such immediate release of glucose and thus
glucosamine.
have less need for highly branched polysaccharides.
The repeating unit in a

glycosaminoglycan
from Wikipedia
70
Hyaluronic Acid
To the tune of Rudolph the Red-Nosed Reindeer
Glycosaminoglycans
Yet another category of polysaccharides are the
glycosaminoglycans
Hyaluronic Acid
Acting almost magically
Placed just beneath the kneecap
Lubricating the debris
Better than joint replacement

Simple as 1-2-3
If it can stop the aching
You will get to keep your knee
When the pain is getting bad

Try not to be sad
Just go out and have a talk
With your orthopedic doc
Beg him to use the needle

To not do so would be a crime
Hyaluronic acid
Workin where the sun dont shine
(also called
mucopolysaccharides),
some examples of which
include keratan sulfate,
heparin, hyaluronic acid
(right), and chondroitin
sulfate. The
polysaccharide
Monomeric unit of chondroitin sulfate.

Chemical structure of one unit in a
chondroitin sulfate chain.
Chondroitin-4-sulfate: R1 = H;
R2 = SO3H; R3 = H.
Chondroitin-6-sulfate: R1 = SO3H;
R2, R3 = H.
compounds are linked to

proteins, but dier from glycoproteins in having a much larger
contingent of sugar residues and, further, the sugars are
considerably more chemically modified. Each of them contains a
repeating unit of a disaccharide that contains at least one
negatively charged residue. The result is a polyanionic substance
that, in its interactions with water, makes for a slimy feel.
Glycosaminoglycans are found in snot, and in synovial fluid,
which lubricates joints. Heparin is a glycosaminoglycan that
H Acid
helps to prevent blood from clotting.
Lipids and
Membranes
Lipids are a broad class of

molecules that all share the

Lipids and Membranes.
71
characteristic that they have at least a portion of them that is

hydrophobic. The class of molecules includes fats, oils (and their
substituent fatty acids), steroids, fat-soluble vitamins,
prostaglandins, glycerophospholipids, sphingolipids, and others.
Interestingly, each one of these can be derived ultimately from
acetyl-CoA.
Fatty Acids
Arguably, the most important lipids in our cells are the fatty acids,
because they are components of all of the other lipids, except
some of the steroids and fat-soluble vitamins. Consisting of a
carboxyl group linked to a long aliphatic tail, fatty acids are
described as either saturated (no double bonds) or unsaturated
(one or more double bonds). Fatty acids with more than one
double bond are described as polyunsaturated. Increasing the
amount of unsaturated fatty acids (and the amount of
Fatty Acids
72
unsaturation in a given fatty acid) in a fat
essential if they must be in the diet (cant be
decreases its melting temperature. This is
synthesized by the organism). Animals, including
also a factor in membrane fluidity. If the
humans, cannot synthesize fatty acids with double
melting temperature of a fat is decreased
bonds beyond position delta 9, so linoleic and
suciently so that it is a liquid at room
linolenic acids are considered essential in these
temperature, it is referred to as an oil. It is
organisms.
worth noting that organisms like fish, which
In animal cells, fats are the primary energy storage
live in cool environments, have fats with
forms. They are also known as triacylglycerols,
more unsaturation. This is why fish oil is a
since they consist of a glycerol molecule esterified
rich source of polyunsaturated fatty acids.
to three fatty acids. Fats are synthesized by
Biochemically, the double bonds found in
replacing the phosphate on phosphatidic acid
fatty acids are predominantly in the cis
with a fatty acid. Fats are stored in the body in
configuration. So-called trans fats arise as a
specialized cells known as adipocytes. Enzymes
chemical by-product of partial
known as lipases release fatty acids from fats by
hydrogenation of vegetable oil (small
hydrolysis reactions. Of the various lipases acting
amounts of trans fats also occur naturally).
on fat, the one that acts first, triacylglycerol lipase,
In humans, consumption of trans fats raises
Fat Hydrolysis
low density lipoprotein (LDL) levels and
is regulated hormonally.
lowers high density lipoprotein (HDL) levels. Each is thought to
Membrane Lipids
contribute to the risk of developing coronary artery disease. The
The predominant lipids found in membranes are
most common fatty acids in our body include palmitate, stearate,
glycerophospholipids (phosphoglycerides) and sphingolipids.
oleate, linolenate, linoleate, and arachidonate. Fatty acids are
The former are related to fats structurally as both are derived from
numbered by two completely dierent schemes. The delta
phosphatidic acid. Phosphatidic acid is a simple
numbering scheme has the carboxyl group as #1, whereas the
glycerophospholipid that is usually converted into phosphatidyl
omega number scheme starts at the other end of the fatty acid
compounds. These are made by esterifying various groups, such
with the methyl group as #1. Fatty acids are described as
as ethanolamine, serine, choline, inositol, and others to the

73
phosphate. All of these compounds form lipid bilayers in

aqueous solution, due to
the amphiphilic nature of
their structure.
Though structurally similar
to glycerophospholipids,
sphingolipids are
synthesized completely
independently of them,
starting with palmitic acid
and the amino acid serine.
The figure on the right
shows the structure of
several sphingolipids. LIke
the glycerophospholipids,
sphingolipids are
The Structures of Several Sphingolipids

74
unsaturation and short fatty acids will favor lower Tm values.

Interestingly, cholesterol does not change the Tm value, but
instead widens the transition range between frozen and fluid
forms of the membrane.
Lipid Bilayers
The membrane around cells contains many components,
including cholesterol, proteins, glycolipids, glycerophospholipids
and
sphingolipids.
The last two of
Cholesterol in a Lipid Bilayer
these will, in
water, form
amphiphilic, but unlike them, they may have simple (in
what is called
cerebrosides) or complex (in gangliosides) carbohydrates
a lipid bilayer,
attached at one end. Most sphingolipids, except sphingomyelin,
which serves
do not contain phosphate.
as a boundary
Steroids, such as cholesterol are also found in membranes.

Cholesterol, in particular, may play an important role in membrane
fluidity. Membranes can be thought of a being more frozen or
more fluid. Fluidity is important for cellular membranes. When
heated, membranes move from a more frozen character to that
of a more fluid one as the temperature rises. The mid-point of
this transition, referred to as the Tm, is influenced by the fatty
acid composition of the lipid bilayer compounds. Longer and
more saturated fatty acids will favor higher Tm values, whereas
for the cell that

is largely
impermeable
to the
movement of
most materials
across it. With
the notable
exceptions of
water, carbon
Lipid bilayer structures

75
dioxide, carbon monoxide, and

oxygen, most polar/ionic
compounds require transport
proteins to help them to
eciently navigate across the
bilayer. The orderly movement of
these compounds is critical for
the cell to be able to 1) get food
for energy; 2) export materials; 3)
maintain osmotic balance; 4)
create gradients for secondary
Types of Membrane Proteins
transport; 5) provide
electromotive force for nerve signaling; and 6) store energy in
into several categories. Integral membrane proteins are
electrochemical gradients for ATP production (oxidative
embedded in the membrane and project through both sides of
phosphorylation or photosynthesis). In some cases, energy is
the lipid bilayer. Peripheral membrane proteins are embedded in
required to move the substances (active transport). In other
or tightly associated with part of the bilayer, but do not project
cases, no external energy is required and they move by diusion
completely through both sides. Associated membrane proteins
through specific cellular channels.
are found near membranes, but may not be embedded in them.
The spontaneous ability of these compounds to form lipid

bilayers is exploited in the formation of artificial membranous
structures called liposomes. Liposomes have some uses in
delivering their contents into cells via membrane fusion.
Membrane Proteins
Other significant components of cellular
membranes include proteins. We can put them
Their association may arise as a result of interaction with other

proteins or molecules in the lipid bilayer. Anchored membrane
proteins are not themselves embedded in the lipid bilayer, but
instead are attached to a molecule (typically a fatty acid) that is
embedded in the membrane.

Membrane Transport.
The geometry of the lipid bilayer is such that is

hydrophobic on its interior and hydrophilic on the
exterior. Such properties also dictate the amino acid
76
side chains of
Cells have hundreds of
proteins that
membrane proteins
interact with the
and the protein
bilayer. For most
composition of a
membrane
membrane varies with
proteins, the
its function and
polar amino
location. Mitochondrial
acids are found
membranes are among
where the protein
the most densely
projects through
packed with proteins.
the bilayer
The plasma membrane
(interacting with
has a large number of
aqueous/polar
integral proteins
substances) and
involved in
the non-polar
communicating
amino acids are
information across the
embedded within
membrane (signaling)
the non-polar
or in transporting
portion of the
materials into the cell.
bilayer
containing the
Sodium-Potassium ATPase
fatty acid tails.
Membrane
Transport
Materials, such as food and waste must be moved across a cells

Glycolipids and glycoproteins play important roles in cellular
lipid bilayer. There are two means of accomplishing this - passive
identification. Blood types, for example, dier from each other in
processes and active processes. Passive processes have as
the structure of the carbohydrate chains projecting out from the
their sole driving force the process of diusion. In these systems,
surface of the glycoprotein in their membranes.
molecules always move from a higher concentration to a lower

77
concentration. These can occur directly
cell and potassium ions into the cell. The protein,
across a membrane (water, oxygen, carbon
which is described as an anti-port (molecules moved
dioxide, and carbon monoxide) or through
in opposite directions across the membrane) uses
special transport proteins (glucose transport
the energy of ATP to create ion gradients that are
proteins of red blood cells, for example). In
important both in maintaining cellular osmotic
each case, no cellular energy is expended in
pressure and (in nerve cells) for creating the ion
the movement of the molecules. On the other
gradients necessary for signal transmission. The
hand, active processes require energy to
transport system moves three atoms of sodium out
accomplish such transport. A common energy
of the cell and two atoms of potassium into the cell
source is ATP (see Na+/K+ ATPase), but many
for each ATP hydrolyzed.
other energy sources are employed. For

example, the sodium-glucose transporter uses
Bacteriorhodopsin
a sodium gradient as a force for actively
An interesting integral membrane protein is
transporting glucose into a cell. Thus, it is
bacteriorhodopsin. The protein has three identical
important to know that not all active transport
polypeptide chains, each rotated by 120 degrees
uses ATP energy. Proteins, such as the
relative to the others. Each chain has seven
sodium-glucose transporter that move two

molecules in the same direction across the
Bacteriorhodopsin
membrane are called symporters (also called
transmembrane alpha helices and contains one

molecule of retinal (Vitamin A) buried deep within
each cavity (shown in purple in lower figure at left).
synporters). If the action of a protein in moving ions across a
Vitamin A is light sensitive and isomerizes rapidly between a cis
membrane results in a change in charge, the protein is described
and a trans form in the presence of light. The changing
as electrogenic and if there is no change in charge the protein is
conformation of the vitamin A is used to transport protons
described as elecro-neutral.
through the protein and out of the bacterium, creating a proton

gradient across the cell membrane, which is used ultimately to
Na+/K+ ATPase
make ATP. It is not too dicult to imagine engineering an
Another important integral membrane protein is the Na+/K+
organism (say a transparent fish) to contain bacteriorhodopsin in
ATPase (previous page), which transports sodium ions out of the
its mitochondrial inner membrane. When light is shone upon it,

78
the bacteriorhodopsin could be used to generate a proton
modifies prothrombin to increase its anity for calcium, allowing
gradient (much like electron transport does) and power oxidative
it to be positioned closer to the site of a wound.
phosphorylation. Such a fish would be partly photosynthetic in

that it would be deriving energy from light, but would dier from
plants in being unable to assimilate carbon dioxide in a series of
dark reactions.
Fat-Soluble Vitamins
Other lipids of note include the fat-soluble vitamins - A, D, E, and
K. Vitamin A comes in three primary chemical forms, retinol
(storage in liver), retinal (role in vision), and retinoic acid (roles in
growth and development). Vitamin D (cholecalciferol) plays
important roles in the intestinal absorption of calcium and
phosphate and thus in healthy bones. Derived from ultimately
from cholesterol, the compound
can be synthesized in a reaction
catalyzed by ultraviolet light.
Top to Bottom - Vitamins E, K, and A
Vitamin E (tocopherol) is the

vitamin about which the least is
known. It consists of a group of
Jump to Chapter
eight fat-soluble compounds of

which the alpha-isomer has the
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
most biological activity. Vitamin K

(the name comes from the German
for coagulation vitamin) is essential
for blood clotting. It is used as a
co-factor for the enzyme that
Vitamin D
79
Chapter 4
Catalysis
In living systems, speed is everything. Providing

the reaction speeds necessary to support life are
the catalysts, mostly in the form of enzymes.
Catalysis
Introduction
Introduction
Activation Energy
General Mechanisms of Action
If there is a magical component to life, an argument can surely be made for it
Substrate Binding
being catalysis. Thanks to catalysis, reactions that could take hundreds of years
Enzyme Flexibility
to complete in the real world, occur in seconds in the presence of a catalyst.
Active Site
Chymotrypsin
Chemical catalysts, like platinum, speed reactions, but enzymes (which are simply
Enzyme Parameters
super-catalysts with a twist) put chemical catalysts to shame. To understand
VMAX & KCAT
KM
enzymatic catalysis, we must first understand energy. In Chapter 2, we noted the

tendency for processes to move in the direction of lower energy. Chemical
Perfect Enzymes
reactions follow this universal trend, but they often have a barrier in place that
Lineweaver-Burk Plots
Enzyme Inhibition

Competitive Inhibition
No Eect On VMAX
Increased KM
Non-Competitive Inhibition
Uncompetitive Inhibition
Suicide Inhibition
Control of Enzymes

Allosterism
Covalent Control of Enzymes
Other Controls of Enzymes
Ribozymes
81
must be overcome. The secret to catalytic action is reducing the

magnitude of that barrier, as we shall see.
Activation Energy
Another feature to note
about catalyzed reactions is Click HERE, HERE, and HERE for

Kevins Catalytic Mechanism
the reduced energy barrier
(also called the activation
lectures on YouTube
The figure below schematically depicts the energy changes that
energy or free energy of
occur during the progression of a simple reaction. In the figure,
activation) to reach the transition state of the catalyzed reaction.
the energy dierences during the reaction are compared for a
This is the second important point about catalyzed reactions
catalyzed (plot on the right) and an uncatalyzed reaction (plot on
catalysts work by lowering activation energies of reactions and
the left). Notice that the reactants start at the same energy level
thus molecules more easily reach the energy necessary to get to
for both conditions and that the products end at the same energy
the point where the reaction occurs. Note that these reactions
for both as well. Thus, the dierence in energy between the
are reversible. The extent to which they will proceed is a function
energy of the ending compounds and the starting compounds is
of the size of the energy dierence between the product and
the same in both
reactant states. The lower
cases. This is the
the energy of the products
first important rule to
compared to the reactants,
understand any kind
the larger the percentage of
of catalysis
molecules that will be
catalysts do not
present as products at
change the overall
equilibrium. At equilibrium,
energy of a reaction.
of course, no change in
Given enough time, a
concentration of reactants
non-catalyzed
and products occurs
reaction will get to
because at this point, the
the same equilibrium
forward and reverse
as a catalyzed one.
reaction rates are the same.
82
mechanism of action of an enzyme and a chemical catalyst is that
General Mechanisms of Action

As noted above, enzymes are orders of magnitude more eective
(faster) than chemical catalysts. The secret of their success lies
in a fundamental dierence in their mechanisms
an enzyme has binding sites that not only grab the substrate
(molecule involved in the reaction being catalyzed), but also place
it in a position to be electronically induced to react, either within
itself or with another substrate. The enzyme
of action. Every chemistry student has had

hammered into their heads the fact that a
Click HERE, HERE, HERE, and
catalyst speeds a reaction without being
HERE for Kevins enzyme
consumed by it. In other words, the catalyst
catalysis lectures on YouTube
ends up after a reaction just the way it started so

it can catalyze other reactions, as well. Enzymes share this
property, but in the middle, during the catalytic action, an enzyme
itself may play a role in the electronic induction

or the induction may occur as a result of
substrates being placed in very close proximity
to each other. Chemical catalysts have no such
ability to bind substrates and are dependent upon
them colliding in the right orientation at or near their surfaces.
is transiently changed. Such changes may be subtle electronic

ones or more significant covalent
modifications. It is also important to
recognize that enzymes are not fixed, rigid
structures, but rather are flexible. Flexibility
allows movement and movement facilitates
alteration of electronic environments
necessary for catalysis. Enzymes are, thus,
much more ecient than rigid chemical
catalysts as a result of their abilities to
facilitate the changes necessary to optimize
the catalytic process.
Substrate Binding
Mechanism of Induced Fit
Another important dierence between the

83
The Way They Work

To the tune of The
Way We Were
Enzymes
Mighty powerhouse peptides
Cause reactions to go faster
In the cells insides
Tiny substrates
Bring about an induced fit
Enzyme structure is affect-ed
By what binds to it
Can it be that its just simple zen?

How the enzymes activate
If they bind effector, will they go
To an R-State, T-State?
Folding
Gives the mechanistic might
To three-D arrangement
Of the active site
Enzymes
Have a bias they cant hide
Hydrophobic side chains are
Mostly found inside
So its the structure

For celebrating
Whenever theres debating
The way they work
Enzyme Flexibility
As mentioned earlier, a dierence between an enzyme and
a chemical catalyst is that an enzyme is flexible. Its slight
changes in shape (often arising from the binding of the
substrate itself) help to position substrates for reaction
after they bind. These changes in shape are explained, in
part, by Koshlands Induced Fit Model of Catalysis,
which illustrates that not only do enzymes change
substrates, but that substrates also transiently change
enzymes. At the end of the catalysis, the enzyme is
returned to its original state. Enzyme flexibility also is
important for control of enzyme activity. Two distinct
structures are typically described the T (tight) state, which
is a lower activity state and the R (relaxed) state, which has
greater activity.
Active Site
Reactions in enzymes are catalyzed at a specific location
known as the active site. Substrate binding sites are
located in close physical proximity to the active site and
oriented to provide access for the relevant portion of the
molecule to the electronic environment of the enzyme
where catalysis is initiated.
Recorded by Liz Bacon and David Simmons

84
chymotrypsin has a hydrophobic hole in which the substrate is
Chymotrypsin
Consider the mechanism of catalysis of the enzyme known as
chymotrypsin. Found in our digestive system, chymotrypsins
catalytic action is cleaving peptide bonds in proteins and it uses
the side chain of a serine in its mechanism of catalysis. Many
other protein-cutting enzymes employ a very similar mechanism
and they are known collectively as serine proteases. As a
protease, it acts fairly specifically, cutting not all peptide bonds,
bound. Preferred substrates will include amino acid side chains

that are hydrophobic, like
phenylalanine. If an ionized side
chain, like that of glutamic acid
binds in the S1 pocket, it will
quickly exit, much like water would
avoid an oily interior. When the
proper substrate binds, it stays and
but only those that are adjacent to
its presence induces an ever so
specific amino acids in the
slight shift in the shape of the
protein. One of the amino acids it
enzyme. This subtle shape change
cuts adjacent to is phenylalanine.
on the binding of the proper
The enzymes action occurs in
substrate starts the steps of the
two phases a fast phase that
catalysis and is the reason that the
occurs first and a slower phase
enzyme shows specificity for
that follows. The enzyme has a
cutting at specific enzyme positions
substrate binding site that
in the target protein. Only amino
includes a region of the enzyme
acids with the side chains that
known as the S1 pocket. Let us
interact well with the S1 pocket
step through the mechanism by
start the catalytic wheels turning.
which chymotrypsin cuts adjacent

to phenylalanine.
The slight changes in shape of the

enzyme upon binding of the proper
Serine Protease Mechanism
The process starts with the
substrate cause changes in the
binding of the substrate in the S1
positioning of three amino acids
pocket. The S1 pocket in
(aspartic acid, histidine, and serine)
Serine Protease
Mechanism
85
The Serine Protease Song

To the tune of Blackbird
Substrate floating in the cells insides
Enzyme snags it with its binding site
It supplies
Shuffling of electrons in the act to catalyze
Proteases of the serine kind
Break up peptide bonds in rapid time
Fast and slow
Steps in breaking bonds are mechanisms you should know
Asp his - ser
Bonds beware
Inside the S1 pocket substrate sits
Alkoxides
Break peptides
Nucleophiles give bonds the fits
Peptide one exits easily
But water has to let the other flee
Bound not free
Cuz the enzymes linked to it in mechanism three
When its gone the enzymes free to catalyze you see
When its gone the enzymes free to catalyze you see
in the active site known as the catalytic triad, during the second
step of the catalytic action. The shift of the negatively charged
aspartic acid towards the electron rich histidine ring favors the
abstraction of a proton by the histidine from the hydroxyl group
on the side chain of serine, resulting in production of a very
reactive alkoxide ion in the active site. Since the active site at
this point also contains the polypeptide chain positioned with the
phenylalanine side chain embedded in the S1 pocket, the
alkoxide ion performs a nucleophilic attack on the peptide bond
on the carboxyl side of phenylalanine sitting in the active site.
This reaction, which is the third step of catalysis, breaks the bond
and causes two things to happen. First, one end of the original
polypeptide is freed and exits the active site. The second is that
the end containing the phenylalanine is covalently linked to the
oxygen of the serine side chain. At this point we have completed
the first (fast) phase of the catalysis.
The second phase of the catalysis by chymotrypsin is slower. It
requires that the covalent bond between phenylalanine and
serines oxygen be broken so the peptide can be released and the
enzyme can return to its original state. The process starts with
entry of water into the active site. Water is attacked in a fashion
similar to that of the serine side chain in the first phase, creating a
reactive hydroxyl group that performs a nucleophilic attack on the
phenylalanine-serine bond, releasing it and replacing the proton
on serine. The second peptide is released in the process and the

reaction is complete with the enzyme back in its original state.
86
Enzyme Parameters
Scientists spend a considerable amount of time characterizing
enzymes. To understand how they do this and what the
characterizations tell us, we must first understand a few
parameters. Imagine I wished to study the reaction catalyzed by
an enzyme I have just isolated. I would be interested to
amount of time and then I would measure the amount of product

contained in each tube. For each reaction, I would determine the
velocity of the reaction as the concentration of product found in
each tube divided by the time. I would then plot the data on a
graph using velocity on the Y-axis and the concentration of
substrate on the X-axis.
understand how fast the
Typically, I would generate a curve like that
enzyme works and how
shown at the left. Notice how the velocity
much anity the enzyme
increase is almost linear in the tubes with the
has for its substrate(s).
lowest amounts of substrate. This indicates

that substrate is limiting and the enzyme
To perform this analysis, I
converts it into product as soon as it can
would perform the
bind it. As the substrate concentration
following experiment. Into
increases, however, the velocity of the
20 dierent tubes, I would
reaction in tubes with higher substrate
put enzyme buer (to keep
concentration ceases to increase linearly and
the enzyme stable), the
instead begins to flatten out, indicating that
same amount of enzyme,
as the substrate concentration gets higher
and then a dierent
and higher, the enzyme has a harder time
amount of substrate in
keeping up to convert the substrate to
each tube, ranging from
product. What is happening is the enzyme is
tiny amounts in the first
becoming saturated with substrate at higher
tubes to very large
concentrations of the latter. Not surprisingly,
amounts in the last tubes.
when the enzyme becomes completely
I would let the reaction

proceed for a fixed, short
saturated with substrate, it will not have to
wait for substrate to diuse to it and will

87
would be necessary to use the same amounts of enzyme in the

dierent reactions they catalyze. It is desirable to have a
measure of velocity that is independent of enzyme
concentration. For this, we define the value Kcat, also known as
the turnover number. Mathematically,
Kcat = Vmax /[Enzyme]
To determine Kcat, one must obviously know the Vmax at a
particular concentration of enzyme, but the beauty of the term is
that it is a measure of velocity independent of enzyme
concentration, thanks to the term in the denominator. Kcat is thus
a constant for an enzyme under given conditions. The units of
Kcat are time-1. An example would be 35/second. This would
mean that each molecule of enzyme is catalyzing the formation
of 35 molecules of product every second. While that might
seem like a high value, there are enzymes known (carbonic
therefore be operating at maximum velocity.
Vmax and Kcat

On a plot of Velocity versus Substrate Concentration (V vs. [S]),
the maximum velocity (known as Vmax) is the value on the Y axis
that the curve asymptotically approaches. It should be noted that
the value of Vmax depends on the amount of enzyme used in a
reaction. Double the amount of enzyme, double the Vmax. If one
wanted to compare the velocities of two dierent enzymes, it
anhydrase, for example) that have Kcat values of 106/second.

This astonishing number illustrates clearly why enzymes seem
almost magical in their action.
KM
Another parameter of an
enzyme that is useful is
Click HERE, HERE, HERE, and

HERE for Kevins Enzyme
Catalysis lectures on YouTube
known as KM, the Michaelis

constant. What it measures, in simple terms, is the anity an
enzyme has for its substrate. Anities of enzymes for substrates
88
vary considerably, so knowing KM helps us to understand how
Movement of substrate by diusion in water has a fixed rate and
well an enzyme is suited to the substrate being used.
that limitation ultimately determines how fast the enzyme can
Measurement of KM depends on the measurement of Vmax. On a
work. In a macroscopic world analogy, factories cant make
V vs. [S] plot, KM is determined as the x value that gives Vmax /2.
products faster than suppliers can deliver materials. It is safe to
A common mistake students make in describing Vmax is saying

that KM = Vmax /2. This is, of course not true. KM is a substrate
say for a perfect enzyme that the only limit it has is the rate of
substrate diusion in water.
concentration and is the amount of substrate it takes for an

enyzme to reach Vmax /2. On the other hand Vmax /2 is a velocity
and is nothing more than that. The value of KM is inversely related
to the anity of the enzyme for its substrate. High values of KM
correspond to low enzyme anity for substrate (it takes more
substrate to get to Vmax). Low KM values for an enzyme
correspond to high anity for substrate.
Perfect Enzymes
Now, if we think about what an ideal enzyme might be, it would
be one that has a very high velocity and a very high anity for its
substrate. That is, it wouldnt take much substrate to get to Vmax/
2 and the Kcat would be very high. Such enzymes would have
values of Kcat / KM that are maximum. Interestingly, there are
several enzymes that have this property and their maximal values
are all approximately the same. Such enzymes are referred to as
being perfect because they have reached the maximum
possible value. Why should there be a maximum possible value
of Kcat / KM? The answer is that movement of substrate to the
enzyme becomes the limiting factor for perfect enzymes.
Avoidance of Formation of an Unstable Intermediate in

Triose Phosphate Isomerase
89
Given the magic of enzymes alluded to earlier, it might seem
glycolysis (figure on previous page). The enzyme appears to have
that all enzymes should have evolved to be perfect. There are
been selected for this ability because at lower velocities, there is
very good reasons why most of them have not. Speed can be a
breakdown of an unstable enediol intermediate that then readily
dangerous thing. The faster a reaction proceeds in catalysis by
forms methyl glyoxal, a cytotoxic compound. Speeding up the
an enzyme, the harder it is to control. As we all know from
reaction provides less opportunity for the unstable intermediate to
learning to drive, speeding causes accident. Just as drivers need
accumulate and fewer undesirable byproducts are made.
to have speed limits for operating automobiles, so too must cells

exert some control on the throttle of their enzymes. In view of
Lineweaver-Burk Plots
this, one might wonder then why any cells have evolved any
The study of enzyme kinetics is typically the most math intensive
enzymes to perfection. There is no single answer to the question,
component of biochemistry and one of the most daunting
but a common one is illustrated by the perfect enzyme known as
aspects of the subject for many students. Although attempts are
triose phosphate isomerase (TPI), which catalyzes a reaction in
made to simplify the mathematical considerations, sometimes

they only serve to confuse or frustrate students. Such is the case
with modified enzyme plots, such as Lineweaver-Burk (left).
Indeed, when presented by professors as simply another thing to
memorize, who can blame students? In reality, both of these
plots are aimed at simplifying the determination of parameters,
such as KM and Vmax. In making either of these modified plots, it
is important to recognize that the same data is used as in making
a V vs. [S] plot. The data are simply manipulated to make the
plotting easier.
For a LineWeaver-Burk, the manipulation is using the reciprocal of
the values of both the velocity and the substrate concentration.
The inverted values are then plotted on a graph as 1/V vs. 1/[S].
Because of these inversions, Lineweaver-Burk plots are
A Lineweaver-Burk Plot of Enzyme Kinetics
commonly referred to as double-reciprocal plots. As can be

90
Two Types of Enzyme Inhibition
seen at left, the value of KM on a Lineweaver Burk plot is easily
discuss four types of enzyme inhibition competitive, non-
determined as the negative reciprocal of the x-intercept , whereas
competitive, uncompetitive, and suicide. Of these, the first three
the Vmax is the inverse of the yintercept. Other related manipulation

of kinetic data include Eadie-Hofstee
diagrams, which plot V vs V/[S] and
give Vmax as the Y-axis intercept with
the slope of the line being - KM.
Enzyme Inhibition
Inhibition of specific enzymes by
drugs can be medically useful.
Understanding the mechanisms of
enzyme inhibition is therefore of
considerable importance. We will
91
types are reversible. The last one is not.
Probably the easiest type of enzyme inhibition to
understand is competitive inhibition and it is the
one most commonly exploited pharmaceutically.
Molecules that are competitive inhibitors of
enzymes resemble one of the normal substrates
of an enzyme. An example is methotrexate,
which resembles the folate substrate of the
enzyme dihydrofolate reductase (DHFR). This
enzyme normally catalyzes the reduction of
folate, an important reaction in the metabolism of
nucleotides. When the drug methotrexate is
present, some of the enzyme binds to it instead
of to folate and during the time methotrexate is
bound, the enzyme is inactive and unable to bind
folate. Thus, the enzyme is inhibited. Notably,
the binding site on DHFR for methotrexate is the
Enzymes
To the tune of Downtown
Reactions alone
Could starve your cells to the bone
Thank God we all produce
Enzymes
Catalytic action won't run wild - don't get hysteric

Cells can throttle pathways with an enzyme allosteric
You know it's true
So when an effector fits
It will just rearrange
all the sub-u-nits
Inside an
ENZYME!
Flipping from R to T
ENZYME!
Slow catalytically
ENZYME!
No change in Delta G
(Enzyme, enzyme)
Units arrange
To make the chemicals change
Because you always use
Enzymes
Sometimes mechanisms run like they are at the races

Witness the Kcat of the carbonic anhydrases
How do they work?

Inside of the active site
It just grabs onto a substrate
and squeezes it tight
In an
ENZYME!
CAT-al-y-sis
In an
ENZYME!
V versus S
In an
ENZYME!
All of this working for you
(Enzyme, enzyme)
You should relax

When seeking out the Vmax though
There are many steps
Enzymes
Lineweaver Burk
Can save a scientist work
With just two intercepts
Enzymes
Plotting all the data from kinetic exploration

Lets you match a line into a best fitting equation
Energy peaks
Are what an enzyme defeats
In its catalysis
Enzymes
Here's what you do

Both axes are inverted then
You can determine Vmax and
Establish Km
for your
ENZYMES!
Sterically holding tight
ENZYMES!
Substrates positioned right
ENZYMES!
Inside the active site
Enzymes (Enzymes, enzymes, enzymes)
Transition state
Is what an enzyme does great
And you should all know this
Enzymes
active site, the same place that folate would

normally bind. As a result, methotrexate
competes with folate for binding to the enzyme.
The more methotrexate there is, the more

eectively it competes with folate for the
enzymes active site. Conversely, the more folate
there is, the less of an eect methotrexate has on
Recorded by Barbara and Neal Gladstone

92
the enzyme because folate outcompetes it.
Increased KM
Note that the apparent KM of the enzyme for the substrate
No Eect on Vmax
How do we study competitive inhibition? It is typically done as
follows. First one performs a set of V vs. [S] reactions without
inhibitor (20 or so tubes, with buer and constant amounts of
increases (-1/KM gets closer to zero - red line above) when the
inhibitor is present, thus illustrating the better competition of the
inhibitor at lower substrate concentrations. It may not be obvious
why we call the changed KM the
enzyme, varying amounts of substrate,

equal reaction times). V vs. [S] is
plotted, as well as 1/V vs. 1/[S], if
desired. Next, a second set of
reactions is performed in the same
manner as before, except that a fixed
amount of the methotrexate inhibitor is
added to each tube. At low
concentrations of substrate, the
inhibitor competes for the enzyme
apparent KM of the enzyme. The

For inhibition, here are rules
To give to students in the schools
Non-competers muddy facts
And drop the value of Vmax
Competers, everyone should know
Will make the KM values grow
Uncompetition makes them think
Since both KM and Vmax shrink
And suicide covalently
Stops enzymes irreversibly
the substrate outcompetes it, due to its higher concentration
actually change the enzymes anity

for the folate substrate. It only
appears to do so. This is because of
the way that competitive inhibition
works. When the competitive inhibitor
binds the enzyme, it is eectively
taken out of action. Inactive enzymes
have NO anity for substrate and no
eectively, but at high concentrations

of substrate, the inhibitor will have a much reduced eect, since
reason is that the inhibitor doesnt
activity either. We cant measure KM for

an inactive enzyme.
(remember that the inhibitor is at fixed concentration).
The enzyme molecules that are not bound by methotrexate can,
Graphically, the results of these experiments are shown above.
in fact, bind folate and are active. Methotrexate has no eect on
Notice that at high substrate concentrations, the competitive
them and their KM values are unchanged. Why then, does KM
inhibitor has essentially no eect, causing the Vmax for the enzyme
appear higher in the presence of a competitive inhibitor? The
to remain unchanged. To reiterate, this is due to the fact that at
reason is that the competitive inhibitor is reducing the amount of
high substrate concentrations, the inhibitor doesnt compete well.
active enzyme at lower concentrations of substrate. When the
However, at lower substrate concentrations it does.
amount of enzyme is reduced, one must have more substrate to
93
supply the reduced amount of enzyme suciently to get to

Vmax/2.
It is worth noting that in competitive inhibition, the percentage of
inactive enzyme changes drastically over the range of [S] values
used. To start, at low [S] values, the greatest percentage of the
enzyme is inhibited. At high [S], no significant percentage of
enzyme is inhibited. This is not always the case, as we shall see
in non-competitive inhibition.
Non-Competitive Inhibition
A second type of inhibition employs inhibitors that do not
resemble the substrate and bind not to the active site, but rather
to a separate site on the enzyme (rectangular site below). The
eect of binding a non-competitive inhibitor is significantly
dierent from binding a competitive inhibitor because there is no
competition. In the case of competitive inhibition, the eect of
the inhibitor could be reduced and eventually overwhelmed with
increasing amounts of substrate. This was because increasing
substrate made increasing percentages of the enzyme active.
With non-competitive inhibition, increasing the amount of
substrate has no eect on the percentage of enzyme that is
active. Indeed, in non-competitive inhibition, the percentage of
enzyme inhibited remains the same through all ranges of [S].
Non-competitive inhibition
typical experiment at every substrate concentration used The

eect of this inhibition is shown above. As you can see, Vmax is
This means, then, that non-competitive inhibition eectively
reduced in non-competitive inhibition compared to uninhibited
reduces the amount of enzyme by the same fixed amount in a
reactions. This makes sense if we remember that Vmax is

94
dependent on the amount of enzyme present. Reducing the
uncompetitive inhibition both decreases Vmax and increases an
amount of enzyme present reduces Vmax. In competitive
enzymes anity for its substrate.
inhibition, this doesnt occur detectably, because at high

substrate concentrations, there is essentially 100% of the enzyme
Suicide Inhibition
active and the Vmax appears not to change. Additionally, KM for
In contrast to the first three types of inhibition, which involve
non-competitively inhibited reactions does not change from that
reversible binding of the inhibitor to the enzyme, suicide inhibition
of uninhibited reactions. This is because, as noted previously,
is irreversible because the inhibitor becomes covalently bound to
one can only measure the KM of active enzymes and KM is a
the enzyme during the inhibition and thus cannot be removed.
constant for a given enzyme.
Suicide inhibition rather closely resembles competitive inhibition
Uncompetitive Inhibition
because the inhibitor generally resembles the substrate and binds

to the active site of the enzyme. The primary dierence is that the
A third type of enzymatic inhibition is that of uncompetitive
suicide inhibitor is chemically reactive in the active site and
inhibition, which has the odd property of a reduced Vmax as well
makes a bond with it that precludes its removal. Such a
as a reduced KM. The explanation for these seemingly odd
mechanism is that
results is rooted in the fact that the uncompetitive inhibitor binds
employed by penicillin
only to the enzyme-substrate (ES) complex. The inhibitor-bound
(right), which covalently
complex forms mostly under concentrations of high substrate and
links to the bacterial
the ES-I complex cannot release product while the inhibitor is
enzyme, D-D
bound, thus explaining the reduced Vmax.
transpeptidase and stops
The reduced KM is a bit harder to conceptualize. The answer lies

in the fact that the inhibitor-bound complex eectively reduces
the concentration of the ES complex. By Le Chateliers Principle,
a shift occurs to form additional ES complex, resulting in less free
enzyme and more enzyme in the forms ES and ESI (ES with
inhibitor). Decreases in free enzyme correspond to an enzyme
with greater anity for its substrate. Thus, paradoxically,
it from functioning. Since
Penicillin
the normal function of the

enzyme is to make a bond necessary for the peptido-glycan
complex of the bacterial cell wall, the cell wall cannot properly
form and bacteria cannot reproduce. If one were to measure the
kinetics of suicide inhibitors under conditions where there was
more enzyme than inhibitor, they would resemble non-competitive
95
inhibitions kinetics because both involve reducing the amount of
reductase, which catalyzes an important reaction in the pathway
active enzyme by a fixed amount in a set of reactions.
leading to the synthesis of cholesterol. Binding of cholesterol to

the enzyme reduces the enzymes activity
Control of Enzymes
significantly. Cholesterol is not a substrate for
It is appropriate that we talk at this point
the enzyme, but, notably, is the end-product
about mechanisms cells use to control
of the pathway that HMG-CoA catalyzes a
enzymes. There are four general
reaction in. When enzymes are inhibited by
methods that are employed. They
an end-product of the pathway in which they
include 1) allosterism; 2) covalent
participate, they are said to be feedback
modification; 3) access to substrate;
inhibited.
and 4) control of enzyme synthesis/
Feedback inhibition always operates by
breakdown. Some enzymes are
allosterism and further, provides important
controlled by more than one of these
and ecient control of an entire pathway. By
methods.
inhibiting an early enzyme in a pathway, the

flow of materials for the entire pathway is
Allosterism
stopped or reduced, assuming there are not
The term allosterism refers to the fact
alternate supply methods. In the cholesterol
that the activity of certain enzymes can
biosynthesis pathway, stopping this one
be aected by the binding of small
enzyme has the eect of shutting o (or at
molecules to the enzyme. In allostery,
least slowing down) the entire pathway.
the molecules that are binding are nonsubstrate molecules that bind at a place
Another excellent example is the enzyme
on the enzyme other than the active

Click HERE, HERE, and HERE for
site.
An excellent example of allosteric
control is the regulation of HMG-CoA
Kinetics of Catalysis by ATCase, an Allosterically

Regulated Enzyme
Kevins Enzyme Regulation

lectures on YouTube
96
aspartate transcarbamoylase (ATCase),
Catalyze
which catalyzes an early reaction in the
To the tune of Close to You

My enzymes
Truly are inclined
To convert
Things they bind
Turn the key
Covalently
Cat-a-lyze
Penicillins action stops

Peptidoglycan cross-links in
Bacterial cell walls in awesome ways
Beta lactam rings reactive site
Starts bonding with D-D-transpeptidase
So there are
Several enzyme states
Counteract
-ing substrates
Now you see
Blocking the key
Regulates
How do cells
Regulate these roles?
Allo-ster
-ic controls
Two forms, you see
States R and T
Mod-u-late
Cat-a-lysts
Have to be controlled
Some get slowed
Put on hold
It's sublime
How the enzymes
(slow) Cat-a-lyze
Competing inhibition keeps

The substrates from the active site
They raise Km, but leave Vmax and shirk
While the non-competers bind elsewhere
And lift the plot made on Lineweaver-Burk
Other ways
Enzymes can be blocked
When things bind
Then get locked
Stuck not free
Tied to the key
Su-i-cide
It's sublime
How the enzymes
(slow) Cat-a-lyze
ahhhhhhhhhhhhhhhhhhh - cat-a-lyze
synthesis of pyrimidine nucleotides. This

enzyme has two allosteric eectors, ATP
and CTP, that are not substrates and that
bind at a regulatory site on the enzyme that
is apart from the catalytic, active site. CTP,
which is the end-product of the pathway, is
a feedback inhibitor of the enzyme. ATP, on
the other hand, acts to activate the enzyme
when it binds to it.
Allosterically, regulation of these enzymes
works by inducing dierent physical states
(shapes, as it were) that aect their ability to
bind to substrate. When an enzyme is
inhibited by binding an eector, it is
converted to the T (also called tight) state,
it has a reduced anity for substrate and it
is through this means that the reaction is
slowed. On the other hand, when an
enzyme is activated by eector binding, it
converts to the R (relaxed) state and binds

substrate much more readily. When no
eector is present, the enzyme may be in a
Recorded by Barbara and Neal Gladstone
mixture of T and R state. The V vs. S plot of

allosteric enzymes resembles the oxygen
97
binding curve of hemoglobin (see HERE). Even though
a wound. Similarly, removal of fibrin clots is also controlled by a
hemoglobin is not an enzyme and is thus not catalyzing a
zymogen (plasminogen), since random clot removal would also
reaction, the similarity of the plots is not coincidental. In both
be hazardous.
cases, the binding of an external molecule is being measured

directly by the hemoglobin plot and indirectly by the enzyme plot,
since substrate binding is a factor in enzyme reaction velocity.
Another common mechanism for control of enzyme activity by

covalent modification is phosphorylation. The phosphorylation of
enzymes (on the side chains of serine, threonine or tyrosine
Covalent Control of Enzymes
residues) is carried out by protein kinases. Enzymes activated by
Some enzymes are synthesized in a completely inactive form and
phosphorylation can be regulated by the addition of phosphate
their activation requires covalent bonds in them to be cleaved.
groups by kinases or their removal by phosphatases.
Such inactive forms of enzymes are called zymogens. Examples

include the proteins involved in blood clotting and proteolytic
Other Enzyme Control Mechanisms
enzymes of the digestive system, such as trypsin, chymotrypsin,
Other means of controlling enzymes relate to access to substrate
and others. The zymogenic forms of these enzymes are known
(substrate-level control) and control of enzyme synthesis.
as trypsinogen and chymotrypsinogen, respectively.
Hexokinase is an enzyme that is largely regulated by availability of
Synthesizing some enzymes in an inactive form makes very good
its substrate, glucose. When glucose concentration is low, the
sense when an enyzmes activity might be harmful to the tissue
product of the enzymes catalysis, glucose-6-phosphate,
where they are being made. For example, the painful condition
accumulates and inhibits the enzymes function.
known as pancreatitis arises when digestive enzymes made in the

pancreas are activated too soon and end up attacking the
pancreas.
Blood clotting involves polymerization of a protein known as
Regulation of enzymes by controlling their synthesis is covered

later in the book in the discussion relating to control of gene
expression.
fibrin. Since random formation of fibrin is extremely hazardous
Ribozymes
(heart attack/stroke), the body synthesizes fibrin as a zymogen
Proteins do not have a monopoly on acting as biological
(fibrinogen) and its activation results from a cascade of
catalysts. Certain RNA molecules are also capable of speeding
activations of proteases that arise when a signal is received from
reactions. The most famous of these molecules was discovered

98
Interactive 4.1
other RNAs have been found.

Ribozymes, however, are not rarities of nature. The proteinmaking ribosomes of cells are essentially giant ribozymes. The
23S rRNA of the prokaryotic ribosome and the 28S rRNA of the
eukaryotic ribosome catalyze the formation of peptide bonds.
Ribozymes are also important in our understanding of the
evolution of life on Earth. They have been shown to be capable
via selection to evolve self-replication. Indeed, ribozymes actually
answer a chicken/egg dilemma - which came first, enzymes that
do the work of the cell or nucleic acids that carry the information
required to produce the enzymes. As both carriers of genetic
information and catalysts, ribozymes are likely both the chicken
and the egg in the origin of life.
Hexokinase - Not Bound to Substrate
by Tom Cech in the early 1980s

Studying excision of an intron in
Tetrahymena, Cech was puzzled at his
inability to find any proteins catalyzing
the process. Ultimately, the catalysis
was recognized as coming from the
intron itself. It was a self-splicing RNA
and since then, many other examples
of catalytic RNAs capable of cutting
Ribozyme Catalytic Action

from Wikipedia
99
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
100
Chapter 5
Flow of
Genetic
Information
As the cells so-called blueprint, DNA must be
copied to pass on to new cells and its integrity
safeguarded. The information in the DNA must also
be accessed and transcribed to make the RNA
instructions that direct the synthesis of proteins.
Flow of Genetic Information

DNA Replication
DNA Repair
DNA Replication
The only way to make new cells is by the division of pre-existing cells. This means
Post-Replicative Mismatch Repair
that all organisms depend on cell division for their continued existence. DNA, as
Systems to Repair Damage to DNA
you know, carries the genetic information that each cell needs. Each time a cell
Nucleotide Excision Repair
Base Excision Repair
Transcription
divides, all of its DNA must be copied faithfully so that a copy of this information
can be passed on to the daughter cell. This process is called DNA replication.
Before examining the actual process of DNA replication, it is useful to think about
what it takes to accomplish this task successfully.
Regulation of Transcription
RNA Processing
Translation
Consider the challenges facing a cell in this process:

The sheer number of nucleotides to be copied is enormous: e.g., in human cells,
on the order of several billion.
A double-helical parental DNA molecule must be unwound to expose single
strands of DNA that can serve as templates for the synthesis of new DNA strands.
This unwinding must be accomplished without
introducing significant topological distortion into
the molecule.
The unwound single strands of DNA must be kept
from coming back together long enough for the
See Kevins YouTube lectures

on DNA Replication and
Repair HERE, HERE, HERE,
and HERE
new strands to be synthesized.

102
DNA polymerases cannot begin synthesis
involved are dierent in bacteria and
of a new DNA strand de novo and require a
eukaryotes, it is useful to understand the
free 3' OH to which they can add DNA
basic considerations that are relevant in all
nucleotides.
cells, before attempting to address the
DNA polymerases can only extend a
details of each system. A generalized
strand in the 5' to 3' direction. The 5' to 3'
account of the steps in DNA replication is
extension of both new strands at a single
presented below, focused on the
replication fork means that one of the
challenges mentioned above.
strands is made in pieces.
The sheer number of nucleotides to be
The use of RNA primers requires that the
copied is enormous.
RNA nucleotides must be removed and

replaced with DNA nucleotides and the
For example, in human cells, the number
resulting DNA fragments must be joined.
of nucleotides to be copied is on the order
Ensuring accuracy in the copying of so
of several billion. Even in bacteria, the
much information.
number is in the millions. Cells, whether
With this in mind, we can begin to examine

how cells deal with each of these
bacterial or eukaryotic, have to replicate all
challenges. Our understanding of the

process of DNA replication is derived from studies using bacteria,
yeast, and other systems, such as Xenopus eggs. These
investigations have revealed that DNA replication is carried out by
of their DNA before they can divide. In

cells like our own, the vast amount of DNA
is broken up into many chromosomes, each of which is
composed of a linear strand of DNA. In cells like those of E. coli,
there is a single circular chromosome.
the action of a large number of proteins that act together as a
In either situation, DNA replication is initiated at sites called
complex protein machine called the replisome. Numerous
origins of replication. These are regions of the DNA molecule that
proteins involved in replication have been identified and
are recognized by special origin recognition proteins that bind the
characterized, including multiple dierent DNA polymerases in
DNA. The binding of these proteins helps open up a region of
both prokaryotes and eukaryotes. Although the specific proteins
single-stranded DNA where the synthesis of new DNA can begin.

103
In the case of E. coli, there is a single origin of

replication on its circular chromosome. In eukaryotic
cells, there may be many thousands of origins of
replication, with each chromosome having hundreds.
DNA replication is thus initiated at multiple points along
each chromosome in eukaryotes as shown in the figure
at the right. Electron micrographs of replicating DNA
from eukaryotic cells show many replication bubbles
on a single chromosome.
This makes sense in light of the large amount of DNA that there
is to be copied in cells like our own, where beginning at one
end of each chromosome and replicating all the way through to
the other end from a single origin would simply take too long.
This is despite the fact that the DNA polymerases in human
cells are capable of building new DNA strands at the very
respectable rate of about 50 nucleotides per second!
A double-helical parental molecule must be unwound to
expose single strands of DNA that can serve as templates for
the synthesis of new DNA strands.
Once a small region of the DNA is opened up at each origin of
replication, the DNA helix must be unwound to allow replication
to proceed. How are the strands of the parental DNA double
helix separated?
104

and HERE
The unwinding of the DNA helix
The unwound single strands of DNA must be kept from coming
requires the action of an enzyme
back together long enough for the new strands to be
called helicase. Helicase uses
synthesized.
the energy released when ATP is

hydrolyzed to unwind the DNA
helix. Note that each replication bubble is made up of two

replication forks that "move" or open up, in opposite directions.
At each replication fork, the parental DNA strands must be
unwound to expose new sections of single-stranded template.
This unwinding must be accomplished without introducing
topological distortion into the molecule.
Once the two strands of the parental DNA molecule are

separated, they must be prevented from going back together to
form double-stranded DNA. To ensure that unwound regions of
the parental DNA remain single-stranded and available for
copying, the separated strands of the parental DNA are bound by
many molecules of a protein called single-strand DNA binding
protein (SSB).
DNA
What is the eect of unwinding one region of the double helix?
polymerases
Unwinding the helix locally causes over-winding or topological
cannot begin
distortion of the DNA ahead of the unwound region. The DNA
synthesis of a
ahead of the unwound helix has to rotate, or it will get twisted on
new DNA
itself.
strand de
How is this problem solved? Enzymes called topoisomerases

can relieve the topological stress caused by local unwinding of
the double helix. They do this by cutting the DNA
and allowing the strands to swivel around each other to release
the tension before rejoining the ends. In E. coli, the
topoisomerase that performs this function is called gyrase.
novo and
require a free
3' OH to
which they
can add DNA
nucleotides.
Addition of a Nucleotide to a Growing RNA Chain
Although
single-stranded parental DNA is now available for copying, DNA
polymerases cannot begin synthesis of a complementary strand
105
region with a free 3'OH group to which DNA polymerase can add
the first new DNA nucleotide (see figure on previous page)
Once a primer provides a free 3'OH for extension, other proteins
get into the act. These proteins are involved in loading the DNA
polymerase onto the primed template and help to keep it
attached to the DNA once it's on.
The first of these is the clamp loader. As its name suggests, the
clamp loader helps to load a protein complex called the sliding
Interactive 5.1
de novo. This is because all DNA polymerases can only add new
nucleotides to the 3' end of a pre-existing chain. This means that
some enzyme other than a DNA polymerase must first make a
small region of nucleic acid, complementary to the parental
strand, that can provide a free 3' OH to which DNA polymerase
can add a deoxyribonucleotide.
This task is accomplished by an enzyme called a primase, which
assembles a short stretch of RNA, called the primer, across from
the parental DNA template. This provides a short base-paired
Sliding Clamp Subunit
106
The 5' phosphate on each incoming nucleotide is joined by the

DNA polymerase to the 3' OH on the end of the growing nucleic
clamp onto the DNA at the replication fork. The sliding clamp is
then joined by the DNA Polymerase. The function of the sliding
clamp is to increase the processivity of the DNA polymerase. This
is a fancy way of saying that it keeps the polymerase associated
with the replication fork by preventing it from falling o- in fact,
the sliding clamp has been described as a seat-belt for the DNA
polymerase.
The DNA polymerase is now poised to start synthesis of the new
DNA strand (in E. coli, the primary replicative polymerase is called
DNA polymerase III). As you already know, the synthesis of new
DNA is accomplished by the addition of new nucleotides
complementary to those on the parental strand. DNA polymerase
catalyzes the reaction by which an incoming deoxyribonucleotide
is added onto the 3' end of the previous nucleotide, starting with
the 3'OH on the end of the RNA primer.
Proteins at a DNA Replication Fork
107
acid chain. As we already
it is being made in the same direction that the replication
noted, the new DNA strands
fork is opening up.
are synthesized by the
The synthesis of the other new strand, called the lagging
addition of DNA nucleotides
strand, requires that multiple RNA primers must be laid
to the end of an RNA primer.

The new DNA molecule thus
down and the new DNA be made in many short pieces
has a short piece of RNA at
that are later joined.These short nucleic acid pieces, each
the beginning.
composed of a small stretch of RNA primer and about

1000-2000 DNA nucleotides, are called Okazaki
DNA polymerases can only
fragments, for Reiji Okazaki, the scientist who first
extend a strand in the 5' to
demonstrated their existence.
3' direction. The 5' to 3'

growth of both new strands
The use of RNA primers requires that the RNA
means that one of the
nucleotides must be removed and replaced with DNA
strands is made in pieces.
nucleotides.
We have noted that DNA
We have seen that each newly synthesized piece of DNA
polymerase can only build a
starts out with an RNA primer, eectively making a new
new DNA strand in the 5' to 3'
nucleic acid strand that is part RNA and part DNA. The
direction. We also know that
finished DNA strand cannot be allowed to have pieces of
the two parental strands of
RNA attached.
DNA are antiparallel. This

means that at each replication
So the RNA nucleotides
fork, one new strand, called
are removed and the
the leading strand can be
gaps are filled in with
synthesized continuously in
DNA nucleotides (by
the 5' to 3' direction because
From Wikimedia Commons
DNA polymerase I in E.
and HERE
coli). The DNA pieces

108
Central Dogma Zen (part 1)

To the tune of Those Were the Days
Polymerase, my friend
Starts at the 3 end
It puts a T across from every A
A G across from C
Perfect simplicity
The leading strand is made

in just this way.
Once upon a time a cell decided
The time was ripe for it to split in two
Had to copy cellular instructions
For the daughter cell would need them too.
Bring in a helicase
Unzip the DNAs
To ease the stress a gyrase joins the fray
Strands must be held apart
SSBs do their part
And primase builds a primer RNA.
The lagging strand is made in little pieces
Okazaki fragments, you recall
Pol I fills the gaps that lie between them
Ligase comes in next and joins them all.
Sliding clamp comes in behind clamp loader
dNTPs floating all around
In the wings a replicase is waiting
For the chance to start another round.
Blueprints cant have mistakes
Thats why polymerase
Corrects its work with exonuclease
Proofreading one by one
Till all its work is done
Hurray for D-N-A polymerase!

Lyrics by Indira Rajagopal
109
are then joined together by the enzyme DNA ligase.
polymerase activity but it can also backtrack and remove the last
inserted base because it has a 3' to 5' exonuclease activity (an
The steps outlined above essentially complete the process of
exonuclease is an enzyme that removes bases, one by one, from
DNA replication. The figure on the previous page shows a

replication fork, complete with the associated proteins that form
the replisome.
the ends of nucleic acids). The exonuclease activity of the DNA

polymerase allows it to excise a wrongly inserted base, after
which the polymerase activity inserts the correct base and
Ensuring accuracy in the copying of so much information
proceeds with extending the strand.
How accurate is the copying of information in the DNA by DNA
In other words, DNA polymerase is monitoring its own accuracy
polymerase? As you are aware, changes in DNA sequence
(also termed its fidelity) as it makes new DNA, correcting mistakes
(mutations) can change the amino acid sequence of the encoded
immediately before moving on to add the next base. This
proteins and that this is often, though not always, deleterious to
mechanism, which operates during DNA replication, corrects
the functioning of the organism. When billions of bases in DNA
many errors as they occur, reducing by about 100-fold the
are copied during replication, how do cells ensure that the newly
mistakes made when DNA is copied.
synthesized DNA is a faithful copy of the original information?

DNA polymerases, as we have noted earlier, work fast (averaging
50 bases a second in human cells and up to 20 times faster in E.
coli). Yet, both human and bacterial cells seem to replicate their
DNA quite accurately. This is because of the proof-reading
function of DNA polymerases. The proof-reading function of a
DNA polymerase enables the polymerase to detect
when the wrong base has been inserted across
from a template strand, back up and remove the
mistakenly inserted base. This is possible because
the polymerase is a dual-function enzyme. It can
extend a DNA chain by virtue of its 5' to 3'
Maintaining the Integrity of the Cell's

Information: DNA Repair
In the last section we considered the ways in which cells deal
with the challenges associated with replicating their DNA, a vital
process for all cells. It is evident that if DNA is the master copy of
instructions for an organism, then it is important not to make
mistakes when copying the DNA to pass on to new
See Kevins YouTube lectures cells. Although proofreading by DNA polymerases

greatly increases the accuracy of replication, there
and HERE
are additional mechanisms in cells to further ensure

that newly replicated DNA is a faithful copy of the
original, and also to repair damage to DNA during
110
the normal life of a cell.
Post-Replicative Mismatch
Repair
All DNA suers damage over
We earlier discussed proof-reading by
time, from exposure to
DNA polymerases during replication.
ultraviolet and other radiation,
Does proofreading eliminate all errors
as well as from various
made during replication? No. While
chemicals in the environment.
proof-reading significantly reduces the
Even chemical reactions
error rate, not all mistakes are fixed on
naturally occurring within cells
the fly by DNA polymerases. What
can give rise to compounds that
mechanisms exist to correct the
can damage DNA. As you
replication errors that are missed by
already know, even minor
the proof-reading function of DNA
changes in DNA sequence, such
polymerases?
as point mutations can

sometimes have far-reaching
Errors that slip by proofreading during
consequences. Likewise,
replication can be corrected by a
unrepaired damage caused by
mechanism called mismatch repair.
radiation, environmental
While the error rate of DNA replication
chemicals or even normal
is about one in 107 nucleotides in the
cellular chemistry can interfere
absence of mismatch repair, this is

Mismatch Repair
with the accurate transmission

of information in DNA.
Maintaining the integrity of the
numerous mechanisms that exist to repair mistakes and damage

in DNA.
in 109 nucleotides when mismatch
cell's "blueprint" is of vital importance and this is reflected in the
further reduced a hundred-fold to one

repair is functional.
What are the tasks that a mismatch repair system faces?

It must:
Scan newly made DNA to see if there are any mispaired bases
111
(e.g., a G paired to a T)
important components of the mismatch repair machinery are the
Identify and cut out the region of the mismatch.
proteins MutS, L and H.
Correctly fill in the gap created by the excision of the mismatch

region.
MutS acts to recognize the mismatch, while MutL and MutH are
recruited to the mismatch site by the binding of Mut S, to help
Importantly, the mismatch repair system must have a means to
cut out the region containing the mismatch. A DNA polymerase
distinguish the newly made DNA strand from the template
and ligase fill in the gap and join the ends, respectively.
strand, if replication errors are to be fixed correctly. In other

words, when the
But how does the mismatch repair system distinguish between

the original and the new strands of
mismatch repair
DNA? In bacteria, the existence of
system encounters an
a system that methylates the DNA
A-G mispair, for
at GATC sequences is the solution
example, it must know
to this problem.
whether the A should

be removed and
E.coli has an enzyme that adds
replaced with a C or if
methyl groups on the to adenines in
the G should be
GATC sequences. Newly replicated
removed and replaced

with a T.
Thymine Dimer Formation in UV Light
Mismatch repair has been well studied in bacteria, and the

proteins involved have been identified. Eukaryotes have a
mismatch repair system that repairs not only single base
mismatches but also insertions and deletions.
In bacteria, mismatch repair proteins are encoded by a group of
genes collectively known as the mut genes. Some of the most
DNA lacks this methylation and

thus, can be distinguished from the
template strand, which is
methylated. In the figure on the previous page, the template

strand shown in yellow is methylated at GATC sequences.
The mismatch repair proteins selectively replace the strand
lacking methylation, shown in blue in the figure, thus ensuring
that it is mistakes in the newly made strand that are removed
and replaced. Because methylation is the criterion that enables
112
the mismatch repair system
Chemical reactions within the cell (such as the
to choose the strand that is
deamination of cytosine to give uracil).
repaired, the bacterial
This means the DNA in your cells is vulnerable to damage
mismatch repair system is

described as being methyl-
simply from normal sorts of actions, such as walking
directed.
outdoors, being in trac, or from the chemical

transformations occurring in every cell as part of its
Eukaryotic cells do not use

this mechanism to
Deamination of Cytosine
distinguish the new strand

from the template, and it is not yet understood how the
mismatch repair system in eukaryotes "knows" which strand
to repair.
Systems to Repair Damage to DNA

In the preceding section we discussed mistakes made when
DNA is copied, where the wrong base is inserted during
synthesis of the new strand. But even DNA that is not being
replicated can get damaged or mutated. These sorts of
damage are not associated with DNA replication, rather they
can occur at any time.
What causes damage to DNA?
Some major causes of DNA damage are:
Radiation (e.g., UV rays in sunlight, in tanning booths)
Exposure to damaging chemicals (such as benzopyrene in
car exhaust and cigarette smoke)
Thymine Dimer Removal

113
everyday activities. (Naturally, the
The Three Rs of DNA
damage is much worse in situations
To the tune of Dream of Little Dream of Me
where exposure to radiation or

damaging chemicals is greater, such as
when people repeatedly use tanning beds
or smoke.)
What kinds of damage do these agents
cause? Radiation can cause dierent
kinds of damage to DNA. Sometimes, as
Base pairs they all provide you

Stair steps to form a helix inside you
A pairs with T and G goes with C
Making DNA for me
Such pathways of excision

Cause cells to have to make a decision
Should they go straight ahead with repair
Or take themselves right out of there?
Helicases go unwinding
Unzippering at rates almost blindin
PO-lymerases work night and day
Replicating D-N-A
(Bridge)
Then lastly, theres recombination
Swap strands readily
Crossover homologous regions
Mix them for me
(Bridge)
Proof-reading - the enzymes QC path
rays, two adjacent pyrimidine bases in the Chews back from the 3s
I cant have a G paired with T so
DNA will be cross-linked to form
Repair it please
pyrimidine dimers (note that we are talking
with much of the damage done by UV
about two neighboring pyrimidine bases

on the same strand of DNA). This is
illustrated in the figure on the previous
Chem damage is concern too

Cuz it can cause mutations inside you
When dimers stem from sunlight UV
Fix the DNA for me
This story is complete now

The DNA is fit for gametes now
The three Rs for the DNA shine
Replicate, repair, recombine
(Oh yeah!)
Replicate, repair, recombine
page where two adjacent thymines on a

single DNA strand are cross-linked to
form a thymine dimer. Radiation can
also cause breaks in the DNA
backbone.
Chemicals like benzopyrene can attach
themselves to bases, forming bulky
DNA adducts in which large chemical
Recorded by Liz Bacon and David Simmons

groups are linked to bases in the DNA.

114
The formation of chemical adducts can physically distort the DNA
DNA is joined to the rest of the DNA
helix, making it hard for DNA and RNA polymerases to copy
backbone by the enzyme DNA
those regions of DNA.
ligase. In E. coli, NER is carried out
Chemical reactions occurring within cells can cause cytosines in

DNA to be deaminated to uracil, as shown in the figure above.
Other sorts of damage in this category include the formation of
oxidized bases like 8-oxo-guanine. These do not actually change
the physical structure of the DNA helix, but they can cause
problems because uracil and 8-oxo-guanine pair with dierent
bases than the original cytosine or guanine, leading to mutations
on the next round of replication.
How do cells repair such damage? Cells have several ways to
remove the sorts of damage described above, with excision
repair being a common strategy. Excision repair is a general term
for the cutting out and re-synthesis of the damaged region of the
DNA. There are a couple of varieties of excision repair:
Nucleotide Excision Repair (NER)
by a group of proteins encoded by

the uvrABC genes. As you can see,
NER is similar, in principle, to
mismatch repair. However, in NER,
the distortion of the helix, caused by
the DNA damage, clearly indicates
which strand of the DNA needs to
be removed and replaced.
Base Excision Repair (BER)

BER deals with situations like the
deamination of cytosine to uracil.
As noted earlier, cytosines in DNA
sometimes undergo deamination to
form the base uracil.
Because cytosines pair with
This system fixes damage by chemicals as well as UV damage.
guanines and uracils pair with
As shown in the figure on the previous page, in nucleotide
adenine, the conversion of cytosine
excision repair, the damage is recognized and a cut is made on
to uracil in the DNA would lead to
either side of the damaged region by an enzyme called an
the insertion of an A in the newly
excinuclease (shown in green). A short portion of the DNA strand
replicated strand instead of the G
containing the damage is then removed and a DNA polymerase
that should have gone in across
fills in the gap with the appropriate nucleotides. The newly made
from a C. To prevent this from
Base Excision Repair

115
happening, uracils are removed from DNA by base excision

repair.
In base excision repair, a single base is first removed from the
DNA, followed by removal of a region of the DNA surrounding the
missing base. The gap is then repaired.
The removal of uracil from DNA is accomplished by the enzyme
uracil DNA glycosylase, which breaks the bond between the uracil
and the sugar in the nucleotide.
The removal of the uracil base creates a gap called an
apyrimidinic site (AP site). The presence of the AP site triggers
the activity of an AP endonuclease that cuts the DNA backbone.
A short region of the DNA surrounding the site of the original
uracil is then removed and replaced, as shown in the figure on the
previous page.
116
Transcription
In the preceding sections, we have discussed the replication of
the cell's DNA and the mechanisms by which the integrity of the
genetic information is carefully maintained. What do cells do with
this information? How does the sequence in DNA control what
happens in a cell? If DNA is a giant instruction book containing
all of the cell's "knowledge" that is copied and passed down from
The Central Dogma
generation to generation, what are the instructions for? And how

do cells use these instructions to make what they need?
Dogma, Central
There's a dogma that's central to cells
That says that the DNA tells
The RNAs to
Make proteins for you
And they'd better get moving, or else.
You have learned in

introductory biology courses
that genes, which are
instructions for making
proteins, are made of DNA.
You also know that information
in genes is copied into
temporary instructions called messenger RNAs that direct the

synthesis of specific proteins. This description of flow of
information from DNA to RNA to protein, shown on the previous
page, is often called the Central Dogma of molecular biology and
is a good starting point for an examination of how cells use the
information in DNA.
Watch Kevins YouTube
RNA Structure
Consider that all of the cells in a
lectures on Transcription
multicellular organism have
HERE, HERE, and HERE
arisen by division from a single

117
baby have the same DNA, each dierent cell

type uses a dierent subset of the genes in
that DNA to direct the synthesis of a
distinctive set of RNAs and proteins. The first
step in gene expression is transcription, which
we will examine next.
What is transcription? Transcription is the
process of copying information from DNA
sequences into RNA sequences.
This process is also known as DNAdependent RNA synthesis. When a sequence
of DNA is transcribed, only one of the two
DNA strands is copied into RNA.
fertilized egg and therefore, all have the same DNA. Division of
that original fertilized egg produces, in the case of humans, over a
trillion cells, by the time a baby is produced from that egg (that's
a lot of DNA replication!). Yet, we also know that a baby is not a
giant ball of a trillion identical cells, but has the many dierent
kinds of cells that make up tissues like skin and muscle and bone
and nerves. How did cells that have identical DNA turn out so
dierent?
The answer lies in gene expression, which is the process by
But, apart from copying one, rather than both

strands of DNA, how is transcription dierent
from replication of DNA? DNA replication serves to copy all the
genetic material of the cell and occurs before a cell divides, so
that a full copy of the cell's genetic information can be passed on
to the daughter cell. Transcription, by contrast, copies short
stretches of the coding regions of DNA to make RNA. Dierent
genes may be copied into RNA at dierent times in the cell's life
cycle. RNAs are, so to speak, temporary copies of instructions of
the information in DNA and dierent sets of instructions are
copied for use at dierent times.
which the information in DNA is used. Although all the cells in a

118
is a general term for an enzyme that makes RNA. There are many
dierent RNA polymerases.
Like DNA polymerases, RNA polymerases synthesize new strands
only in the 5' to 3' direction, but because they are making RNA,
they use ribonucleotides (i.e., RNA nucleotides) rather than
deoxyribonucleotides. Ribonucleotides are joined in exactly the
same way as deoxyribonucleotides, which is to say that the 3'OH
of the last nucleotide on the growing chain is joined to the 5'
phosphate on the incoming nucleotide.
One important dierence between DNA polymerases and RNA
T7 RNA Polymerase making RNA (green)
using DNA template (brown)
Cells make several dierent kinds of RNA:

mRNAs that code for proteins
rRNAS that form part of ribosomes
tRNAs that serve as adaptors between mRNA and amino acids
during translation
Micro RNAs that regulate gene expression
Other small RNAs that have a variety of functions.
Building an RNA strand is very similar to building a DNA strand.
This is not surprising, knowing that DNA and RNA are very similar
molecules. What enzyme carries out transcription? Transcription
is catalyzed by the enzyme RNA Polymerase. "RNA polymerase"
polymerases is that the latter do not require a primer to start

making RNA. Once RNA polymerases are in the right place to
start copying DNA, they just begin making RNA by stringing
together RNA nucleotides complementary to the DNA template.
This, of course, brings us to an obvious question- how do RNA
polymerases "know" where to start copying on the DNA? Unlike
the situation in replication, where every nucleotide of the parental
DNA must eventually be copied, transcription, as we have already
noted, only copies selected genes into RNA at any given time.
Consider the challenge here: in a human cell, there are
approximately 6 billion basepairs of DNA. Most of this is noncoding DNA, meaning that it won't need to be transcribed. The
small percentage of the genome that is made up of coding
sequences still amounts to between 20,000 and 30,000 genes in
119
each cell. Of these genes, only a small number will need to be
dependent on the binding of RNA polymerase to the promoter
expressed at any given time.
sequence to begin transcription. If the RNA polymerase and its
What indicates to an RNA polymerase where to start copying
helper proteins do not bind the promoter, the gene cannot be

transcribed and it will therefore, not be
expressed.
What is special about a promoter sequence? In
an eort to answer this question, scientists
looked at many genes and their surrounding
sequences. It makes sense that because the
same RNA polymerase has to bind to many
Promoter Sequences
DNA to make a transcript? Signals in DNA indicate to RNA

polymerase where it should start (and end) transcription. These
signals are special sequences in DNA that are recognized by the
RNA polymerase or by proteins that help RNA polymerase
dierent promoters, the promoters should have

some similarities in their sequences. Sure
enough, common sequence patterns were seen to be present in
many promoters. We will first take a look at prokaryotic
promoters. When prokaryotic genes were examined, the
determine where it should bind the DNA to start transcription. A

DNA sequence at which the RNA polymerase binds to start
transcription is called a promoter.
A promoter is generally situated upstream of the gene that it
controls. What this means is that on the DNA strand that the
gene is on, the promoter sequence is "before" the gene.
Remember that, by convention, DNA sequences are read from 5'
to 3'. So the promoter lies 5' to the start point of transcription.
Also notice that the promoter is said to "control" the gene it is
associated with. This is because expression of the gene is
Promoter Sequence Elements
following features commonly emerged (see figure above):

A transcription start site (this the base in the DNA across from
which the first RNA nucleotide is paired).
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A -10 sequence: this is a 6 bp region
makes RNA. Together, the sigma
centered about 10 bp upstream of the start
subunit and core polymerase make
site. The consensus sequence at this
up what is termed the RNA
position is TATAAT. In other words, if you
polymerase holoenzyme. The
count back from the transcription start site,
sigma subunit of the polymerase
which by convention, is called the +1, the
(shown in brown in the figure) can
sequence found at -10 in the majority of
recognize and bind to the -10 and
promoters studied is TATAAT).
-35 sequences in the promoter,

thus positioning the RNA
A -35 sequence: this is a sequence at about
polymerase (shown in green) at the
35 basepairs upstream from the start of
right place to initiate transcription.
transcription. The consensus sequence at this
Once transcription begins, the core
position is TTGACA.
polymerase and the sigma subunit
What is the significance of these sequences?
separate, with the core polymerase
It turns out that the sequences at -10 and -35
continuing RNA synthesis and the
are recognized and bound by a subunit of
sigma subunit wandering o to
prokaryotic RNA polymerase before
escort another core polymerase
transcription can begin.
molecule to a promoter. The sigma

subunit can be thought of as a sort
The RNA polymerase of E. coli, for example,
of usher that leads the polymerase
has a subunit called the sigma subunit (or
to its "seat" on the promoter.
sigma factor) in addition to the core

polymerase,
As already mentioned, an RNA
which is the
chain, complementary to the DNA
part of the
template, is built by the RNA
enzyme that
polymerase by the joining of the 5'
actually
Transcription Initiation in E. coli
phosphate of an incoming
121
ribonucleotide to the
3'OH on the last
nucleotide of the
growing RNA
strand. How does
the polymerase
know where to
stop? A sequence
of nucleotides
called the
terminator is the
signal to the RNA
polymerase to stop
transcription and
dissociate from the
template.
The Students Guide to a Perfect Transcript
with histones and other

proteins. The "packaging"
of the DNA must therefore
Pol II's so smart, now let me see
The last one is a special case
It makes a transcript, one, two, three,
It moonlights as a helicase
From a billion base pairs can it find
And sends Pol II upon its way
The one promoter it must bind?
To make a brand new RNA.
I need some help, I hear it plead,
How does it do that, you may ask
A second dierence is that
This DNA I cannot read
A phosphate does the crucial task
eukaryotes have multiple
I cannot see where I must start
Added on to CTD,
RNA polymerases, not one
'Til TFIIs have done their part.
That's how the H sets Pol II free.
as in bacterial cells. The
be opened up to allow the

RNA polymerase access to
the template in the region
to be transcribed.
dierent polymerases
When TATA's bound by TBP
And Pol II goes its merry way
transcribe dierent genes.
That sends a signal out, you see
There soon will be some RNA
For example, RNA
Once DNA has made a bend
Then introns must be all excised
polymerase I transcribes
More TFIIs will soon attend.
As RNA is capped and spliced.
the ribosomal RNA genes,
Although the
while RNA polymerase III
process of RNA
When B arrives, it clears a place
Then PolyA polymerase
copies tRNA genes. The
synthesis is the
For RNA polymerase
Will add a couple hundred As
RNA polymerase we will
same in eukaryotes
And F and E soon join the fun
For my own transcript, I can say
focus on most is RNA
as in prokaryotes,
TFIIH, the final one.
That all I want is one more A.
polymerase II, which
there are some

additional issues to
transcribes protein-coding
Verse by Indira Rajagopal
genes to make mRNAs.
keep in mind in eukaryotes. One is that in eukaryotes, the DNA

template exists as chromatin, where the DNA is tightly associated
122
All three eukaryotic RNA polymerases need
RNA polymerase to position
additional proteins to help them get transcription
itself correctly to begin
started. In prokaryotes, RNA polymerase by itself
transcription. (Some
can initiate transcription (remember that the sigma
eukaryotic promoters lack
subunit is a subunit of the prokaryotic RNA
TATA boxes, and have,
polymerase). The additional proteins needed by
instead, other recognition
eukaryotic RNA polymerases are referred to as
sequences to help the RNA
transcription factors. We will see below that there
polymerase find the spot on
are various categories of transcription factors.
the DNA where it spot on the

DNA where it binds and
Finally, in eukaryotic cells, transcription is
initiates transcription.)
separated in space and time from translation.

Transcription happens in the nucleus, and the
We noted earlier that
mRNAs produced are processed further before they
eukaryotic RNA polymerases
are sent into the cytoplasm. Protein synthesis
need additional proteins to
(translation) happens in the cytoplasm. In
bind promoters and start
prokaryotic cells, mRNAs can be translated as they
transcription. What are these
are coming o the DNA template, and because
additional proteins that are
there is no nucleus, transcription and protein
needed to start transcription?
synthesis occur in a single cellular compartment.
General transcription factors

are proteins that help
Like genes in prokaryotes, eukaryotic genes also
eukaryotic RNA polymerases
have promoters. Eukaryotic promoters commonly
find transcription start sites
have a TATA box, a sequence about 25 basepairs
Assembly of the Basal Transcription
upstream of the start of transcription that is

recognized and bound by proteins that help the
Complex and Initiation of Transcription
and initiate RNA synthesis.

We will focus on the
transcription factors that
123

assist RNA polymerase II. These
polymerase from the basal transcription complex and allows it to
transcription factors are named
move forward and begin transcription.
TFIIA, TFIIB and so on (TF=

transcription factor, II=RNA
Regulation of Transcription
polymerase II, and the letters
The processes described above are required whenever any gene
distinguish individual transcription factors).

Transcription in eukaryotes requires the general transcription
factors and the RNA polymerase to form a complex at the TATA
box called the basal transcription complex or transcription
initiation complex. This is the minimum requirement for any gene
to be transcribed. The first step in the formation of this complex
is the binding of the TATA box by a transcription factor called the
is transcribed. But what determines which genes are transcribed

at a given time? What are the molecular switches that turn
transcription on or o? Although there are entire books written on
this one topic, the basic mechanism by which transcription is
regulated depends on highly specific interactions between
transcription regulating proteins and regulatory sequences on
DNA.
TATA Binding Protein or TBP. Binding of the TBP causes the DNA
We know that promoters indicate where transcription begins, but
to bend at this spot and take on a structure that is suitable for the
what determines that a given gene will be transcribed? In
binding of additional transcription factors and RNA polymerase.
addition to the promoter sequences required for transcription
As shown in the figure at left, a number of dierent general
initiation, genes have additional regulatory sequences (sequences
transcription factors, together with RNA polymerase (Pol II) form a
of DNA on the same DNA molecule as the gene) that control
complex at the TATA box.
when a gene is transcribed. Regulatory sequences are bound
The final step in the assembly of the basal transcription complex

is the binding of a general transcription factor called TFIIH. TFIIH
is a multifunctional protein that has helicase activity (i.e., it is
capable of opening up a DNA double helix) as well as kinase
tightly and specifically by transcriptional regulators, proteins that

can recognize DNA sequences and bind to them. The binding of
such proteins to the DNA can regulate transcription by preventing
or increasing transcription from a particular promoter.
activity. The kinase activity of TFIIH adds a phosphate onto the
Let us first consider an example from prokaryotes. In bacteria,
C-terminal domain (CTD) of the RNA polymerase. This
genes are often clustered in groups, such that genes that need to
phosphorylation appears to be the signal that releases the RNA
be expressed at the same time are next to each other and all of
124
How do bacteria achieve this?

Transcription of the lac cluster of genes is
primarily controlled by a repressor protein
that binds to a region of the DNA just
downstream of the -10 sequence of the lac
promoter. Recall that the promoter is where
the RNA polymerase must bind to begin
transcription. The place where the repressor
is bound is called the operator (labeled O in
the figure). When the repressor is bound at
Thegenes
geneslac
lacz,z,lac
lacyyand
andlac
lacaaare
areall
allunder
under the
the control
control of a single
The
single promoter
promoterininthe
thelac
lacoperon
operon
them are controlled as a single unit by the same promoter. The

lac operon, shown below, is one such group of genes that
encode proteins needed for the uptake and breakdown of the
sugar lactose. The three genes of the lac operon, lac z, lac y
and lac a are controlled by a single promoter.
Bacterial cells generally prefer to use glucose for their energy
needs, but if glucose is unavailable, and lactose is present, the
bacteria will take up lactose and break it down for energy.
Since the proteins for taking up and breaking down lactose are
only needed when glucose is absent and lactose is available,
the bacterial cells need a way to express the genes of the lac
operon only under those conditions. At times when lactose is
absent, the cells do not need to express these genes.
Lac Operon Regulation
125
this position, it physically blocks the RNA polymerase from
(Technically, the inducer is allolactose, a molecule made from
transcribing the genes, just as a vehicle blocking your driveway
lactose by the cell, but the principle is the same.)
would prevent you from pulling out.
What makes this an especially eective control system
Obviously, if you want to leave, the vehicle that is
is that the genes of the lac operon encode proteins
blocking your path must be removed. Likewise,
that break down lactose. Turning on these genes
in order for transcription to occur, the repressor

must be removed from the operator to clear the
Regulation HERE, HERE, and requires lactose to be present. Once the lactose is
HERE
broken down, the repressor binds to the operator once
path for RNA polymerase. How is the repressor
more and the lac genes are no longer expressed. This
removed?
allows the genes to be expressed only when they are needed.
When the sugar lactose is present, it binds to the repressor,
But how do glucose levels aect the expression of the lac genes?
changing its conformation
We noted earlier that if glucose
so that it no longer binds
was present, lactose would not be
to the operator. When the
used. A second level of control is
repressor is no longer
exerted by a protein called CAP
bound at the operator, the
that binds to a site adjacent to the
"road-block" in front of
promoter and recruits RNA
the RNA polymerase is
polymerase to bind the lac
removed, permitting the
promoter. When glucose is
transcription of the genes
depleted, there is an increase in
of the lac operon.
levels of cAMP which binds to CAP.

The CAP-cAMP complex then
Because the binding of
binds the CAP site, as shown in the
the lactose induces the
figure. The combination of CAP
expression of the genes in
binding and the lac repressor
the lac operon, lactose is

called an inducer.
Lac Operon Activation
dissociating from the operator

126
when lactose levels are high ensures high levels of transcription
of tryptophan. This is achieved by having a repressor protein that
of the lac operon just when it is most needed. The CAP protein
will bind to the operator only in the presence of tryptophan.
binding may be thought of as a green light for the RNA
Transcription in eukaryotes is also regulated by the binding of
polymerase, while the removal of repressor is like the lifting of a
proteins to specific DNA sequences, but with some dierences
barricade in front of it.
from the simple
When both conditions are
schemes outlined
met, the RNA polymerase
above. For most
transcribes the downstream
eukaryotic genes,
genes.
general
The lac operon we have just
transcription
described is a set of genes
factors and RNA
that are expressed only
polymerase (i.e.,
under the specific
the basal
conditions of glucose
transcription
depletion and lactose
complex) are
availability. Other genes
necessary, but not
may be expressed unless a
sucient, for high
particular condition is met.
levels of
An example of this is the trp
transcription.
operon in bacterial cells,
Enhancer Mechanism
which encodes enzymes

necessary for the synthesis of the amino acid tryptophan. These
genes are expressed at all times, except when tryptophan is
available from the cell's surroundings. This means that these
genes must be prevented from being expressed in the presence
In eukaryotes,
additional
regulatory sequences called enhancers and the proteins that bind

to the enhancers are needed to achieve high levels of
transcription. Enhancers are DNA sequences that regulate the
transcription of genes. Unlike prokaryotic regulatory sequences,
127
enhancers don't need to be next
structure of the chromatin in that region. As we noted earlier, in
to the gene they control. Often
eukaryotes, DNA is packaged with proteins to form chromatin.
Regulation HERE, HERE, and they are many kilobases away on

the DNA. As the name suggests,
HERE
enhancers can enhance
When the DNA is tightly associated with these proteins, it is

dicult to access for transcription. So proteins that can make the
DNA more accessible to the transcription machinery can also play
(increase) transcription of a particular gene.
a role in the extent to which transcription occurs.
How can a DNA sequence far from the gene being transcribed
In addition to enhancers, there are also negative regulatory
aect the level of its transcription?
sequences called silencers. Such regulatory sequences bind to
Enhancers work by binding proteins (transcriptional activators)

that can, in turn, interact with the proteins bound at the promoter.
The enhancer region of the DNA, with its associated
transcriptional activator(s) can come in contact with the basal
transcription complex that is bound at a distant TATA box by
looping of the DNA (previous page). This allows the protein
bound at the enhancer to make contact with the proteins in the
basal transcription complex.
transcriptional repressor proteins.Transcriptional activators and

repressors are modular proteins- they have a part that binds DNA
and a part that activates or represses transcription by interacting
with the basal transcription complex.
RNA Processing
So far, we have looked at the mechanism by which the
information in genes (DNA) is transcribed into RNA. The newly
One way that the transcriptional activator bound to

the enhancer increases the transcription from a
distant promoter is that it increases the frequency
and eciency with which the basal transcription
complex is formed at the promoter.
Another mechanism by which proteins bound at the
enhancer can aect transcription is by recruiting to
the promoter other proteins that can modify the
RNA Splicing
128
made RNA, also known as the primary

transcript (the product of transcription is
known as a transcript) is further processed
before it is functional. Both prokaryotes
and eukaryotes process their ribosomal
and transfer RNAs.
The major
dierence in
RNA
processing,
however,
Processingof
Eukaryotic
Steps
Steps in
in processing
eukaryoticMessenger
messengerRNAs
RNAS
between
containing introns will also therefore have regions that interrupt
prokaryotes and eukaryotes, is in the
the information in the gene. These regions must be removed
processing of messenger RNAs. We
before the mRNA is sent out of the nucleus to be used to direct
will focus on the processing of mRNAs
protein synthesis. The process of removing the introns and
in this discussion. You will recall that in
rejoining the coding sections or exons, of the mRNA, is called
bacterial cells, the mRNA is translated
splicing. Once the mRNA has been capped, spliced and had a
directly as it comes o the DNA
polyA tail added, it is sent from the nucleus into the cytoplasm for
template. In eukaryotic cells, RNA
translation.
synthesis, which occurs in the nucleus,
mRNA Cap Structure
is separated from the protein synthesis
The initial product of transcription of a protein coding gene is
machinery, which is in the cytoplasm.
called the pre-mRNA (or primary transcript). After it has been
In addition, eukaryotic genes have
processed and is ready to be exported from the nucleus, it is
introns, noncoding regions that
called the mature mRNA or processed mRNA.
interrupt the genes coding sequence.

The mRNA copied from genes
129
protects the 5' end of the mRNA from degradation by

nucleases and also helps to position the mRNA correctly on the
ribosomes during protein synthesis.
The 3' end of a eukaryotic mRNA is first trimmed, then an
enzyme called PolyA Polymerase adds a "tail" of about 200 A
nucleotides to the 3' end. There is evidence that the polyA tail
plays a role in ecient translation of the mRNA, as well as in
the stability of the mRNA. The cap and the polyA tail on an
mRNA are also indications that the mRNA is complete (i.e., not
defective).
Introns are removed from the pre-mRNA by the activity of a
complex called the spliceosome. The spliceosome is made up
of proteins and small RNAs that are associated to form proteinRNA enzymes called small nuclear ribonucleoproteins or
Splicing
What are the processing steps for messenger RNAs?

In eukaryotic cells, pre-mRNAs undergo three main processing
steps:
Capping at the 5' end
Addition of a polyA tail at the 3' end. and
Splicing to remove introns
In the capping step of mRNA processing, a 7-methyl guanosine
(shown at left) is added at the 5' end of the mRNA. The cap
Splicing and Protein Diversity

130
snRNPs (pronounced SNURPS). The splicing machinery must be
In the second step, the 3' splice
able to recognize splice junctions (i.e., the end of each exon and
site is cut, and the two exons
the start of the next) in order to correctly cut out the introns and
are joined together, and the
join the exons to make the mature, spliced mRNA.
intron is released.

lectures on Translation HERE,
HERE, and HERE
What signals indicate where an intron starts and ends?
Many pre-mRNAs have a large number of exons that can be
The base sequence at the start (5' or left end, also called the
spliced together in dierent combinations to generate dierent
donor site) of an intron is GU while the sequence at the 3' or right
mature mRNAs. This is called alternative splicing, and allows the
end (a.k.a. acceptor site) is AG. There is also a third important
production of many dierent proteins using relatively few genes,
sequence within the intron, called a branch point, that is
since a single RNA can, by combining dierent exons during
important for splicing.
splicing, create many dierent protein coding messages. Because

of alternative
There are two main steps
splicing, each gene
in splicing:
in our DNA gives

rise, on average, to
In the first step, the premRNA is cut at the 5'
three dierent
splice site (the junction
proteins.
of the 5' exon and the
Once protein
intron). The 5' end of
coding messages
the intron then is joined
have been
to the branch point
processed by
within the intron. This
capping, splicing
generates the lariat-
and addition of a
shaped molecule
poly A tail, they are
characteristic of the
transported out of
splicing process
Coupled Transcription and Translation in Prokaryotes
the nucleus to be
131
activity, known as
a peptidyl
transferase, that
makes the
peptide bonds
that join amino
acids to make a
polypeptide. The
small and large
subunits
assemble on the
mRNA at its
5end to initiate
translation.
The Genetic Code
translated in the cytoplasm.
Translation
Ribosomes
The Codon Song

To the tune of When I'm Sixty Four
Building of proteins, you oughta know

Needs amino A's
Peptide bond catalysis in ribosomes
Triplet bases, three letter codes
Mixing and matching nucleotides
Who is keeping score?
Here is the low down
If you count codons
You'll get sixty four
Got - to - line - up - right
16-S R-N-A and
Shine Dalgarno site
You can make peptides, every size
With the proper code
Start codons positioned
function by
In the P site place
binding to
Initiator t-RNAs
mRNAs and
holding them in a
UGA stops and AUGs go

Who could ask for more?
You know the low down
Translation is the process by which information in mRNAs is used
way that allows
Count up the codons
to direct the synthesis of proteins. As you have learned in
the amino acids
There are sixty four
introductory biology, in eukaryotic cells, this process is carried
encoded by the
out in the cytoplasm of the cell, by large RNA-protein machines
RNA to be joined
called ribosomes. Ribosomes contain ribosomal RNAs (rRNAs)
sequentially to
and proteins. The proteins and rRNAs are organized into two
form a
subunits, a large and a small. The large subunit has an enzymatic
polypeptide.

132
The sequence of
an mRNA directly
specifies the
sequence of

lectures on Translation HERE,
HERE, and HERE
amino acids in the
This ingenious system is used to direct the assembly

of a protein in the same way that you might string
together colored beads in a particular order using
instructions that used symbols like 111 for a red bead,
followed by 222 for a green bead, 333 for yellow, and
protein it encodes. Each amino
so on, till you came to 000, indicating that you should stop
acid in the protein is specified by a
stringing beads.
sequence of 3 bases called a

codon in the mRNA. For example,
the amino acid tryptophan is
tRNA Structure
encoded by the sequence 5UGG3
Interactive 5.2
on an mRNA. Given that there are

4 bases in RNA, the number of
dierent 3-base combinations that are possible is 43, or 64.

There are, however, only 20 amino acids that are used in building
proteins. This discrepancy in the number of possible codons and
the actual number of amino acids they specify is explained by the
fact that the same amino acid may be specified by more than one
codon. In fact, with the exception of the amino acids methionine
and tryptophan, all the other amino acids are encoded by multiple
codons. The figure above shows the codons that are used for
each of the twenty amino acids.
Three of the 64 codons are known as termination or stop codons
and as their name suggests, indicate the end of a protein coding
sequence. The codon for methionine, AUG, is used as the start,
Phenylalanyl-tRNA
or initiation, codon.
133
A tRNA with an amino acid attached to it is

said to be charged. Another region of the
tRNA has a sequence of 3 bases, the
anticodon, that is complementary to the
codon for the amino acid it is carrying.
When the tRNA encounters the codon for its
amino acid on the messenger RNA, the
anticodon will base-pair with the codon, and
mRNA Alignment by Shine-Dalgarno Sequence
the amino acid attached to it will be brought

in to the ribosome to be added on to the
While the ribosomes are literally the protein factories that join
amino acids together using the instructions in mRNAs, another
class of RNA molecules, the transfer RNAs (tRNAs) are also
needed for translation. Transfer RNAs (see figure, left) are small
RNA molecules, about 75-80 nucleotides long, that function to
'interpret' the instructions in the mRNA during protein synthesis.
In terms of the bead analogy above, someone, or something, has
to be able to bring a red bead in when the instructions indicate
111, and a green bead when the instructions say 222.
growing protein chain.

With an idea of the various components necessary for translation
we can now take a look at the process of protein synthesis. The
main steps in the process are similar in prokaryotes and
eukaryotes. As we already noted, ribosomes bind to mRNAs and
facilitate the interaction between the codons in the mRNA and the
anticodons on charged tRNAs.
Unlike a human, who can choose a red bead when 111 is

present in the instructions, neither ribosomes nor tRNAs
can think. The system, therefore, relies, like so many
processes in cells, solely on molecular recognition.
A given transfer RNA is specific for a particular amino acid.
It is linked covalently to this amino acid at its 3' end by an
Shine-Dalgarno Sequences
enzyme called aminoacyl tRNA synthetase. There is an

aminoacyl tRNA synthetase specific for each amino acid.
134
A,P and E Sites in the Ribosome
In bacterial cells, translation is coupled with

transcription and begins even before the mRNA has
been completely synthesized. How does the
ribosome recognize and bind to the mRNA? Many
bacterial mRNAs carry a short purine-rich sequence
known as the Shine-Dalgarno site upstream of the
AUG start codon, as shown in the figure below. This
sequence is recognized and bound by a
complementary sequence in the 16S rRNA that is
part of the small ribosomal subunit as shown above.
Because the Shine-Dalgarno site serves to recruit
and bind the ribosome, it is also referred to as the
Ribosome Binding Site or RBS.
A variation of this process of ribosome assembly
operates in eukaryotic cells. We already know that in
eukaryotic cells, processed mRNAs are sent from the
Initiation of Translation
nucleus to the cytoplasm. The small and large

subunits of ribosomes, each composed of
135
The Ribosome
To the tune of America the Beautiful
O beautiful with R-N-A

That makes the peptide bonds
You hold t-RNA so it
Can pair up with co-dons
The Ribosome! The Ribosome!
Translate m-RNA
Initiate and translocate
From start to U-G-A

characteristic rRNAs and proteins are found in the cytoplasm and

assemble on mRNAs to form complete ribosomes that carry out
translation.
Protein synthesis in eukaryotes starts with the binding of the
small subunit of the ribosome to the 5' end of the mRNA. The
assembly of the translation machinery begins with the binding of
the small ribosomal subunit to the 7-methyl guanosine cap on the
5'end of an mRNA. Meanwhile, the initiator tRNA pairs with the
Elongation of Translation
start codon. (Recall that the start codon is AUG, and codes for
methionine. The initiator tRNA carries the amino acid methionine).
136
Interactive 5.3
called initiation and requires the help of protein factors called

initiation factors.
The second codon of the mRNA is positioned adjacent to the
second site on the ribosome, the A site. This is where the tRNA
carrying the amino acid specified by the second codon binds.
The binding of aminoacyl tRNA to the A-site is mediated by
proteins called elongation factors and requires the input of
energy. Once the appropriate charged tRNAS have "docked" on
the codons by base-pairing between the anticodon on the tRNA
and the codon on the mRNA, the ribosome joins the amino acids
carried by the two tRNAs by making a peptide bond (see figure at
right).
Interestingly, the formation of the peptide bond is catalyzed by a
catalytic RNA (the 23S rRNA in prokaryotes) rather than by a
The Ribosome
The large subunit of the ribosome then joins the complex, which
is now ready to start protein synthesis.
protein enzyme.
This and subsequent steps in the synthesis of the polypeptide are
called the elongation phase of translation. Once the first two
amino acids are linked , the first tRNA dissociates, and moves out
Ribosomes have two sites for binding charged tRNAs, each of
of the P-site and into the E, or Exit site. The second tRNA then
which is positioned to make two adjacent codons on the mRNA
moves into the P-site, vacating the A-site for the tRNA
available for binding by tRNAs. The initiation codon occupies the
corresponding to the next codon.
first of these ribosomal sites, the P-site. The anticodon

complementary to this is on the initiator tRNA, which brings in the
first amino acid of the protein. This initial phase of translation is
The process repeats till the stop codon is in the A-site. At this
point, a release factor binds at the A-site, adds a water molecule
to the polypeptide at the P-site, and releases the completed
137
Central Dogma Zen- Part 2

To the tune of Those Were The Days
An organism's cellular construction
With blueprints for the things they have to do
Requires converting DNA instructions
To ribopolymers, oh yes it's true
The process moves along without much trouble

While making RNA inside the cell
It all occurs inside transcription bubbles
Where bases get linked anti-parallel
Because they've been bestowed

With a genetic code
The RNAs provide the cell with means
To link amino A's
In most directed ways
Inside the protein-making cell machines
mRNA then roams

To find some ribosomes
Subunits large and small bind near the end
The A-U-G's in place
Inside the P site space
Initiation you can comprehend
If "coli" cells don't have galactosidase

And lactose should appear inside its food
The lac repressor leaves the operator
'Cause otherwise metabolism's screwed
The mechanism shifts to elongation

Proceeding by three bases at a stretch
A GTP's required for translocation
Advancing 5 to 3 the whole complex
Polymerase unwinds
The DNAs it binds
Adjacent to the start site where it docks
Unravels A's and T's
With such amazing ease
At the promoter's little TATA box
The process moves anon

Until a stop codon
Arrives and causes movement to suspend
Translation has to cease
A peptide gets released
And we have reached the central dogma's end

138
polypeptide from the ribosome, which itself, then dissociates into

subunits.
As described in Chapter 3, polypeptides made in this way are
then folded into their three dimensional shapes, posttranslationally modified and delivered to the appropriate cellular
compartments to carry out their functions.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
139
Chapter 6
Metabolism I
Depending on your mathematical perspective, life is

the sum or the product of the biochemical reactions
that occur in cells. The collection of these reactions
is known as metabolism. We break the subject into
two broad areas - 1) oxidative/reductive metabolism
and 2) pathways that involve little oxidation/
reduction. This chapter deals with the former.
Section 1
Metabolism I
Definitions
The cost of living is energy and the producers and consumers of energy in the cell
Perspectives
are the chemical reactions known collectively as metabolism. Metabolic
Glycolysis

Intermediates

Reactions

Enzymes/Control

Pyruvate Metabolism
Gluconeogenesis
Citric Acid Cycle
Glyoxylate Pathway
Acetyl-CoA Metabolism
Cholesterol Metabolism
Ketone Body Metabolism
Prostaglandin Synthesis
Fatty Acid Oxidation

Oxidation of Odd-Chain Fatty Acids

Unsaturated Fatty Acid Oxidation

Enzymes of Beta Oxidation

Alpha Oxidation
Fatty Acid Synthesis

Enzymes of Fatty Acid Synthesis

Elongation of Fatty Acids

Desaturation of Fatty Acids
Metabolism of Fat
Connections to Other Pathways
processes are governed by the same laws of energy as the rest of the universe, so
they must be viewed in the light of Gibbs free energy.
For the most part, the drivers of changes in Gibbs free energy are changes in
concentration of reactants and products, but for some reactions, the concentration
changes required to run a reaction in the desired direction are not practical. In
such cases, cells may use alternative strategies, such as energy coupling reactions
(combining an energetically unfavorable reaction with a favorable one, such as the
hydrolysis of ATP) to help drive the unfavorable reaction. In other cases, cells
use alternate pathways around energetically unfavorable reactions.
Definitions
We start by defining a few terms.
Anabolic processes refer to collections
of biochemical reactions that make bigger
molecules from smaller ones. Examples
include the synthesis of fatty acids from
acetyl-CoA, of proteins from amino acids,
A student really ought to know

The ways in which electrons flow
When running catabolically
Removal yields some energy
If anabolic bliss ensues
The tiny charges then reduce
of complex carbohydrates from simple
141
organisms is not made in
Click HERE, HERE, and
directly in these reactions.
HERE for Kevins YouTube
Instead, the electrons released
lectures on Metabolism and
by oxidation are collected by
Glycolysis
electron carriers which donate

them, in the mitochondria, to complexes that make ATP
(ultimately) by oxidative phosphorylation.
Redox Reactions
In our tour of metabolism, we will tackle in this chapter processes

sugars, and of nucleic acids from nucleotides. Just as any
that are the most oxidative/reductive in nature and in the
construction project requires energy, so, too, do anabolic
following chapter those pathways that involve less reduction/
processes require input of energy. Anabolic processes tend to be
oxidation. The aim in this coverage is not to go through the step-
reductive in nature, in contrast to catabolic processes,
by-step reactions of the pathway, but rather to focus on control
which are oxidative. Not all anabolic processes are

reductive, though. Protein synthesis and nucleic acid
synthesis do not involve reduction, though the synthesis of
amino acids and nucleotides does.
Catabolic processes are the primary sources of energy for
heterotrophic organisms and they ultimately power the
anabolic processes. Examples include glycolysis
(breakdown of glucose), the citric acid cycle, and fatty acid
oxidation. Reductive processes require electron sources,
such as NADPH, NADH, or FADH2. Oxidative processes
require electron carriers, such as NAD+, NADP+, or FAD.
Catabolic processes are ultimately the source of ATP
energy in cells, but the vast majority of ATP in heterotrophic
NAD+ / NADH Structures

142
points, interesting
pathways made up of
enzymes, molecules
reactions for breakdown and
common between
synthesis of each of these
pathways, and how the
compounds. The figure at
metabolic pathways meet
left shows such a simple
the organisms needs.
schematic and how the

pathways are not isolated
Perspective
from each other molecular
We can view metabolism
products of one are
at several levels. At the
substrates for another. At a
highest level, we have
deeper level, we can study
nutrients, such as sugars,
individual reactions and
fatty acids and amino
discover the enormous
acids entering cells and
complexity and commonality
carbon dioxide and other
of metabolic reactions.
waste products (such as
In studying metabolism, we
urea) exiting. Cells use
recognize that metabolic
the incoming materials for
pathways are manmade
energy and substance to
concepts with artificial
synthesize sugars,
boundaries. Students
nucleotides, and other
commonly think of the
amino acids as building

blocks for the
carbohydrates, nucleic
molecules in the pathways

Catabolism and Anabolism
acids, fatty compounds,

and proteins necessary for life. As we zoom in, we can imagine
being tied exclusively to

those individual pathways,
but with the exception of reactions that have physical barriers

(such as those occurring within an organelle), metabolic
143
The Reactions of Glycolysis

from Wikipedia
pathways have many common intermediates used in multiple

reactions occurring in the same location at the same time and
thus cannot be ascribed to any one pathway. The best we can do
is understand general directions of pathways in cells.
Glycolysis
The metabolic pathway traditionally covered first in biochemistry
texts is glycolysis and there seems to be no reason to break that
trend here. Glycolysis, which literally means breakdown of
144
Your cells may have a mounting crisis

Should they not go through glyco-lye-sis
No glucose energy releases
Unless its fractured into pieces
sugar, is a
molecules of pyruvate, which, in turn, can be oxidized further in
catabolic process in
citric acid cycle.
which six carbon

sugars (hexoses)
Intermediates
are oxidized and
Glucose and fructose are the sugar funnels serving as entry
broken down into
points to the glycolytic pathway. Other sugars must be converted
pyruvate molecules. The corresponding anabolic pathway by
to either of these forms to be directly metabolized. Some
which glucose is synthesized is termed gluconeogenesis. Both

glycolysis and gluconeogenesis are not major
oxidative/reductive processes by themselves,
with one step in each one involving loss/gain of
electrons, but the product of glycolysis,
pathways, including the Calvin Cycle and the Pentose
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HERE for Kevins YouTube
lectures on Metabolism and
pyruvate, can be completely oxidized to carbon
Glycolysis
dioxide. Indeed, without production of pyruvate

from glucose in glycolysis, a major energy source for the cell is
not available. By contrast, gluconeogenesis can synthesize
Phosphate Pathway (PPP, see below) contain

intermediates in common with glycolysis, so in that
sense, almost any cellular sugar can be metabolized
here. Intermediates of glycolysis that are common to
other pathways include glucose-6-phosphate (PPP,
glycogen metabolism), F6P (PPP), G3P (Calvin, PPP), DHAP (PPP,

glycerol metabolism, Calvin), 3PG (Calvin, PPP), PEP (C4 plant
glucose reductively from very simple materials, such as pyruvate

and acetyl-CoA/glyoxylate (at least in plants). For these reasons
we include these pathways in the red/ox collection.
Glucose is the most abundant hexose in nature and is the one
people typically associate with glycolysis, but fructose (in the
form of fructose-6-phosphate) is metabolized in the cell and
galactose can easily be converted into glucose for catabolism in
the pathway as well. The end metabolic products of the pathway
are two molecules of ATP, two molecules of NADH and two
Step 1 of Glycolysis
145
With the pump thus primed, the pathway proceeds first to split
the F1,6BP into two 3-carbon intermediates. Later the only
oxidation step in the entire pathway occurs. In that reaction,
glyceraldehyde-3-phosphate (G3P) is oxidized and a phosphate
is added, creating 1,3-bisphosphoglycerate (1,3 BPG).
The addition of the phosphate sometimes conceals the oxidation

that occurred. G3P was an aldehyde. 1,3 BGP is an acid
Steps 2 (top) and 4 (bottom) of Glycolysis
esterified to a phosphate. The two phosphates in the tiny 1,3BPG

molecule repel each other and give the molecule high energy. It
metabolism, Calvin), and pyruvate (fermentation, acetyl-CoA
uses this energy to phosphorylate ADP to make ATP.
genesis, amino acid metabolism).
Reactions
The pathway of glycolysis begins with two inputs of energy. First,
glucose gets a phosphate from ATP to make glucose-6phosphate (G6P) and later fructose-6-phosphate (F6P) gets
another phosphate from ATP to make fructose-1,6bisphosphate (F1,6BP).
146
2-PG is converted to phosphoenolpyyruvate (PEP) by removal of

water, creating a very high energy intermediate. Conversion of
PEP to pyruvate is the second substrate level phosphorylation of
glycolysis, creating ATP. There is almost enough energy in PEP to
Since there are two 1,3 BPGs produced for every glucose, the
two ATP produced replenish the two ATPs used to start the cycle.
The synthesis of ATP directly from a metabolic reaction is known
as substrate level phosphorylation (see HERE), though it is not
a significant source of ATP. Glycolysis has two reactions during
which substrate-level phosphorylation occurs.
The transfer of phosphate from 1,3BPG to ATP creates 3phosphoglycerate (3-PG). Conversion of 3-PG to 2-PG occurs
by an important mechanism. An intermediate in the reaction
(catalyzed by phosphoglycerate mutase) is 2,3 BPG. This
intermediate, which is stable, is released with low frequency by
the enzyme instead of being converted to 2-PG. 2,3BPG is
important because it binds to hemoglobin and stimulates release
of oxygen (see HERE). Thus, cells which are metabolizing
glucose rapidly release more 2,3BPG and, as a result, stimulate
release of more oxygen, supporting their needs.
Glycolysis Regulation
147
stimulate production of a second ATP, but
The Sound of Glucose
it is not used. Consequently, the energy
To the tune of A Few of My Favorite Things
is lost as heat. If you wonder why you get

hot when you exercise, the reaction that
converts PEP to pyruvate is a prime
culprit.
Enzymes/Control
Control of glycolysis is unusual for a
metabolic pathway, in that regulation
occurs at three enzymatic points
hexokinase (Glucose <=>G6P),
phosphofructokinase - PFK (F6P <=>
F1,6BP), and pyruvate kinase (PEP <=>
pyruvate).
Glycolysis is regulated in a reciprocal
fashion compared to its corresponding
anabolic pathway, gluconeogenesis.
Reciprocal regulation occurs when the
Aldehyde sugars are always aldoses and

If there's a ketone we call them ketoses
Some will form structures in circular rings
Saccharides do some incredible things
Phosphate plus ADP makes ATP

While giving cells what they need - en-er-gy
Making triphosphate's a situa-shun
Of substrate level phosphoryla-shun
Onto a glucose we add a 'P' to it

ATP energy ought to renew it
Quick rearranging creates F6P
Without requiring input energy
3-B-P-G
2-B-P-G
Lose a water
PEP gets a high energy state
Just to make py-ru-vate
At a high rate
Add a phosphate
With PFK
F1,6BP is made up this way
So we can run and play
So all the glucose gets broken and bent

If there's no oxygen cells must ferment
Pyruvate / lactate our cells hit the wall
Some lucky yeast get to make ethanol
This is the end of your glucose's song
Aldolase breaks it and then it releases

DHAP and a few G3Pieces
These both turn in to 1,3 BPG
Adding electrons onto NAD
Unless you goof up and get it all wrong

Break it, don't make it to yield ATP
You'll save your cells from fu-til-i-ty
same molecule or treatment

(phosphorylation, for example) has
opposite eects on catabolic and

anabolic pathways. Reciprocal regulation
is important when anabolic and
corresponding catabolic pathways are
occurring in the same cellular location.

148
As an example, consider regulation of PFK. It

is activated by several molecules, most
importantly fructose-2,6-bisphosphate
(F2,6BP). This molecule has an inhibitory eect
on the corresponding gluconeogenesis
enzyme, fructose-1,6-bisphosphatase
(F1,6BPase).
For cells a glucose cyclings cost

Is energy in reams
Four ATPs each time is lost
From breaking/making schemes
So use for metabolic heat
To make it warm inside your feet
Else its of no utility
To practice such futility
happens, some of the excess F1,6BP activates

pyruvate kinase, which jump-starts the
conversion of PEP to pyruvate. The resulting
drop in PEP levels has the eect of pulling on
the reactions preceding pyruvate kinase. As a
consequence, the concentrations of G3P and
DHAP fall, helping to move the aldolase
reaction forward.
You might wonder why pyruvate kinase, the last enzyme in the
pathway, is regulated. The answer is simple. Pyruvate kinase
catalyzes the most energetically rich reaction of glycolysis. The
reaction is favored so strongly in the forward direction that cells
must do a two-step around it in the reverse direction when
making glucose. In other words, it takes two enzymes, two
reactions, and two triphosphates to go from pyruvate back to
Pyruvate Metabolism
As noted, pyruvate produced in glycolysis can be oxidized to
acetyl-CoA, which is itself oxidized in the citric acid cycle to
carbon dioxide. That is not the only metabolic fate of pyruvate,
though.
PEP in gluconeogenesis. When cells are needing to make
Pyruvate is a starting point for gluconeogenesis, being
glucose, they cant be sidetracked by having the PEP they have
converted to oxaloacetate in the mitochondrion in the first step.
made in gluconeogenesis be converted directly back to pyruvate
Pyruvate in animals can also be reduced to lactate when oxygen
by pyruvate kinase. Consequently, pyruvate kinase is inhibited

during gluconeogenesis, lest a futile cycle occur. (See HERE)
Another interesting control mechanism called feedforward
activation involves pyruvate kinase. Pyruvate kinase is activated
allosterically by F1,6BP. This molecule is a product of the PFK
reaction and a substrate for the aldolase reaction. It should be
noted that the aldolase reaction is energetically unfavorable (high
+ G), thus allowing F1,6BP to accumulate. When this
Reduction of Pyruvate
149
ethanol from pyruvate under anaerobic conditions, instead of

lactic acid. Thus, fermentation of pyruvate is necessary to keep
glycolysis operating when oxygen is limiting. It is also for these
reasons that brewing of beer (using yeast) involves depletion of
oxygen and muscles low in oxygen produce lactic acid (animals).
Pyruvate is a precursor of alanine which can be easily
synthesized by transfer of a nitrogen from an amine donor, such
as glutamic acid. Pyruvate can also be converted into
oxaloacetate by carboxylation in the process of gluconeogenesis
(see below).
The enzymes involved in pyruvate metabolism include pyruvate
dehydrogenase (makes acetyl-CoA), lactate dehydrogenase
(makes lactate), transaminases (make alanine), and pyruvate
carboxylase (makes oxaloacetate).
Metabolic Redox
is limiting. This reaction, which requires NADH produces NAD+

and is critical for generating the latter molecule to keep the
glyceraldehyde-3-phosphate dehydrogenase reaction of
glycolysis going when there is no oxygen.
Gluconeogenesis
The anabolic counterpart to glycolysis is gluconeogenesis, which
occurs mostly in the cells of the liver and kidney. In seven of the
eleven reactions of gluconeogenesis (starting from pyruvate), the
same enzymes are used as in
Oxygen is necessary for the electron transport system to operate
glycolysis, but the reaction
and this, in turn, is what oxidizes NADH to NAD+. In the absence
directions are reversed. Notably,
of oxygen, thus, an alternative means of making NAD+ is
the G values of these reactions
necessary, or else glycolysis will halt. Bacteria and yeast have
in the cell are typically near zero,
NADH requiring reactions that regenerate NAD+ while producing
meaning their direction can be readily controlled by changing
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Gluconeogenesis
150
Gluconeogenesis
To the tune of Supercalifragelisticexpialidocious
When cells have lots of ATP and NADH too
They strive to store this energy as sugar yes they do
Inside of mitochondria they start with pyruvate
(slow) Carboxylating it to make oxaloacetate
Oh gluconeogenesis is anabolic bliss

Reversing seven mechanisms of glycolysis
To do well on the final students have to learn all this
Gluconeogenesis is anabolic bliss
Oh gluconeogenesis is so exhilarating
Memorizing it can really be exasperating
Liver cells require it so theres no need for debating
Gluconeogenesis is so exhilarating
Oh, glucose, glucose factory

Glucose, glucose factory
Oh, glucose, glucose come to be

Glucose, glucose come to be
The aldolase reaction puts together pieces so

A fructose molecule is made with two phosphates in tow
And one of these gets cleaved off by a fructose phosphatase
(slow) Unless F2,6BP's acting blocking path-a-ways
Oxaloacetate has got to turn to PEP

Employing energy that comes from breaking GTP
From there it goes to make a couple phosphoglycerates
(slow) Exploiting ee-nolase and mutase catalytic traits
Oh gluconeogenesis a pathway to revere

That makes a ton of glucose when it kicks into high gear
The cell's a masterminding metabolic engineer
Gluconeogenesis a pathway to revere
Oh gluconeogenesis is livers specialty

Producing sugar for the body most admirably
Six ATPs per glucose is the needed energy*
Gluconeogenesis is livers specialty
Oh glucose, glucose jubilee

Glucose, glucose jubilee
Oh glucose, glucose joy to me

Glucose, glucose joy to me
From F6P to G6P, that is the final phase

The enzyme catalyzing it is an isomerase
Then G6P drops phosphate and a glucose it becomes
(slow) Inside the tiny endoplasmic-al reticulums
Converting phosphoglycerate to 1,3BPG

Requires a phosphate that includes A-T-P energy
Reduction with electrons gives us all an N-A-D
(slow) And G3Ps isomerized to make D-H-A-P
Oh gluconeogenesis is not so very hard

I know that on the final we will not be caught off guard
Cuz our professor lets us use a filled out index card
Gluconeogenesis is not so very hard

151
substrate and product concentrations.

Directional velocity
Inverts with reciprocity
If glycolysis is flowing
Glucose synthesis awaits
But when the latter is a-going
Sugar breakdown then abate
The three regulated enzymes of

glycolysis all catalyze reactions whose
G values are not close to zero,
making manipulation of reaction
direction non-trivial. Consequently,
cells employ work-around reactions
importance because cells generally need
catalyzed by four dierent enzymes to
to minimize the extent to which paired
favor gluconeogenesis, when
anabolic and catabolic pathways are
appropriate.
occurring simultaneously, lest they waste

energy and make no tangible product
Two of the enzymes (pyruvate
except heat. The mechanisms of
carboxylase and PEP carboxykinase -
controlling these pathways work, in some
PEPCK) catalyze reactions that
ways, in opposite fashions, called
bypass pyruvate kinase. F1,6BPase
reciprocal regulation (see above).
bypasses PFK and G6Pase bypasses

hexokinase. Notably, pyruvate
Besides reciprocal regulation, other
carboxylase and G6Pase are found in
mechanisms help control
the mitochondria and endoplasmic
gluconeogenesis. First, PEPCK is
reticulum, respectively, whereas the
controlled largely at the level of synthesis.
other two are found in the cytoplasm
Overexpression of PEPCK (stimulated by
along with all of the enzymes of
glucagon, glucocorticoids, and cAMP
glycolysis. As a result, all of glycolysis
and inhibited by insulin) causes
and most of gluconeogenesis occurs
symptoms of diabetes. Pyruvate
in the cytoplasm. Controlling these

pathways then becomes of critical
Glycolysis and Gluconeogenesis
carboxylase is sequestered in the

mitochondrion and is sensitive to acetyl152
CoA, which is an allosteric activator. Acetyl-CoA concentrations
cells, so they dump it into the blood. Lactate travels in the blood
increase as the citric acid cycle activity decreases. Glucose-6-
to the liver, which takes it up and reoxidizes it back to pyruvate,
phosphatase is present in low concentrations in many tissues,
catalyzed by the enzyme lactate dehydrogenase. Pyruvate in the
but is found most abundantly and importantly in the major
liver is then converted to glucose by gluconeogenesis. The
gluconeogenic organs the liver and kidney cortex.
glucose thus made by the liver is dumped into the bloodstream

where it is taken up by muscles and used for energy, completing
Cori Cycle
a very important intercellular pathway known as the Cori cycle.
With respect to energy, the liver and muscles act

complementarily. The liver is the major organ in the body for the
Citric Acid Cycle
synthesis of glucose. Muscles are major users of ATP. Actively
The primary catabolic pathway in the body is the citric acid cycle
exercising
because it is here that oxidation to carbon dioxide occurs for
muscles generate
breakdown products of the cells major building blocks - sugars,
lactate as a result
fatty acids, amino acids. The pathway is cyclic (next page) and
of running
thus, doesnt really have a starting or ending point. All of the
glycolysis faster
reactions occur in the mitochondrion, though one enzyme is
than the blood
embedded in the organelles membrane. As needs change, cells
can deliver
may use a subset of the reactions of the cycle to produce a
oxygen during
desired molecule rather than to run the entire cycle (see below for
periods of heavy
examples).
exercise. As a
Focusing on the pathway itself, the traditional point to start
consequence,
discussion is addition of acetyl-CoA to oxaloacetate (OAA) to
the muscles go
form citrate. Acetyl-CoA for the pathway can come from a variety
anaerobic and
of sources. They include
produce lactate.
pyruvate oxidation (from
This lactate is of
no use to muscle
glycolysis and amino acid
metabolism), fatty acid
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YouTube lectures on the Citric
Acid and Glyoxylate Cycles
153
oxidation, and amino acid metabolism. The reaction joining

it to OAA is catalyzed by citrate synthase and the G is
fairly negative. This, in turn, helps to pull the reaction
preceding it in the cycle (catalyzed by malate
dehydrogenase).
In the next reaction, citrate is isomerized to isocitrate by
action of the enzyme called aconitase. Isocitrate is a
branch point in plants and bacteria for the glyoxylate
cycle. Oxidative decarboxylation of isocitrate by isocitrate
dehydrogenase produces the first NADH and yields alphaketoglutarate. This five carbon intermediate is a branch
point for synthesis of glutamate. In addition, glutamate can
also be made easily into this citric acid cycle intermediate.
Decarboxylation of alpha-ketoglutarate yields succinylCoA and is catalyzed by alpha-ketoglutarate
dehydrogenase. This enzyme is structurally very similar to
pyruvate dehydrogenase and employs the same five
coenzymes NAD, FAD, CoASH, TPP, and lipoic acid.
The remainder of the citric acid cycle involves conversion
of the four carbon succinyl-CoA into oxaloacetate.
Succinyl-CoA is a branch point for the synthesis of heme.
Succinyl-CoA is converted to succinate in a reaction
catalyzed by succinyl-CoA synthetase (named for the
Citric Acid Cycle Reactions
reverse reaction) and a GTP is produced, as well the only

substrate level phosphorylation in the cycle. The energy for
154
the synthesis of the GTP
FADH2 it gains in the reaction to coenzyme Q. The product of
comes from hydrolysis
the reaction, fumarate gains a water across its trans double bond
of the high energy
in the next reaction, catalyzed by fumarase to form malate.
thioester bond between
Fumarate is also a byproduct of nucleotide metabolism and of the
Aconitase is picky
Binds substrates specially
Creating isocitrate
Which has no symmetry
succinate and the CoA.
urea cycle. Malate is important also for transporting electrons
Evidence for the high
across membranes in the malate aspartate shuttle and in
energy of a thioester
Then CO2 gets lost from it

Released in the next phase
The secret weapon - Isocitrate
Dehydrogenase
bond is also evident in
ferrying carbon dioxide in C4 plants.
I love my citrate synthase

It really is first rate
Adds O-A-A to Ac-Co-A
Producing a citrate
The alpha KD-H is next

It gets my admiration
For clipping CO2 in one more
Decarboxylation
Succ-CoA synthetase steps up
Reacting most absurd
Its named for a catalysis
That really goes backward
Suc -CIN-ate de-hyd-ROG-en-ase
Pulls H from succinate
Creating FADH2
As well as fumarate
The fumarate gains water
OH-configured L
The fumarases product?
Some malate for the cell
With the last oxidation
Malate de-hyd-ROG-en-ase
Expels its two creations
N-A-D-H / O-A-A
the citrate synthase
Conversion of malate to OAA is a rare biological oxidation that
reaction, which is also
has a G with a positive value. The reaction product includes
very energetically
NADH and the reaction is pulled by the energetically favorable
favorable. Succinate is
conversion of OAA to citrate in what was described above as the
also produced by
first reaction of the cycle. OAA intersects other important
metabolism of odd-
pathways, including amino acid metabolism (readily converted to
chain fatty acids (see
aspartic acid), transamination (nitrogen movement) and
below).
gluconeogenesis.
Oxidation of succinate
Glyoxylate Pathway
occurs in the next step,

catalyzed by succinate
dehydrogenase. This
interesting enzyme both
catalyzes this reaction
and participates in the
electron transport
system, funneling
electrons from the
A pathway related to
the Citric Acid
Cycle (CAC) is the
glyoxylate
pathway (right).
This pathway,
which overlaps all
of the non-
Im thinking I could lose some weight

If I could make glyoxylate
Combined with acetyl-CoA
Malate would then form OAA
The excess OAA in turn
Would give more glucose to be burned
Converting fat to glucose, see
Expends it glycolytically
155
malate (catalyzed by malate synthase). Malate can, in turn, be

oxidized to oxaloacetate.
It is at this point that the pathways contrast with the CAC is
apparent. After one turn of the CAC, a single oxaloacetate is
produced and it balances the single one used in the first reaction
of the cycle. Thus, in the CAC, no net production of oxaloacetate
is realized. By contrast, at the end of a turn of the glyoxylate
cycle, two oxaloacetates are produced, starting with one. The
extra oxaloacetate can then be used to make other molecules,
including glucose in gluconeogenesis.
Because animals do not run the glyoxylate cycle, they cannot
produce glucose from acetyl-CoA in net amounts, but plants and
bacteria can. As a result, these organisms can turn acetyl-CoA
from fat into glucose, while animals cant. Bypassing the
The Glyoxylate Cycle
decarboxylation reactions of the CAC does not operate in

animals, because they lack two enzymes necessary for the
pathway isocitrate lyase and malate synthase. Isocitrate lyase
catalyzes the conversion of isocitrate into succinate and
glyoxylate. Because of this, all six carbons of the CAC survive
and do not end up as carbon dioxide.
decarboxylations (and substrate level phosphorylation) has its

costs, however. Each turn of the glyoxylate cycle produces one
FADH2 and one NADH instead of the three NADHs, one FADH2,
and one GTP made in each turn of the CAC.
Acetyl-CoA Metabolism
Acetyl-CoA is one of the most connected metabolites in
biochemistry, appearing in fatty acid oxidation/reduction,
Succinate continues through the remaining reactions of the CAC
pyruvate oxidation, the citric acid cycle, amino acid anabolism/
to produce oxaloacetate. Glyoxylate combines with another
catabolism, ketone body metabolism, steroid/bile acid synthesis,
acetyl-CoA (one acetyl-CoA was used to start the cycle) to create
and (by extension from fatty acid metabolism) prostaglandin

156
synthesis. Most of these pathways will be dealt with separately.

Here we will cover the last three.
The pathways for ketone body synthesis and cholesterol
biosynthesis overlap at the beginning. Each of these starts by
combining two acetyl-CoAs together to make acetoacetyl-CoA.
Not coincidentally, that is the next to last product of oxidation of
fatty acids with even numbers of carbons (see below). In fact, the
enzyme that catalyzes the joining is the same as the one that
catalyzes its breakage in fatty acid oxidation thiolase. Thus,
these pathways start by reversing the last step of the last round
of fatty acid oxidation. Both pathways also include addition of
two more carbons from a third acetyl-CoA to form HydroxyMethyl-Glutaryl-CoA, or HMG-CoA, as it is more commonly
known. It is at this point that the two pathways diverge.
Cholesterol Metabolism
The cholesterol biosynthesis pathway is a long one and it requires
significant amounts of reductive and ATP energy, which is why it
is included here. Cholesterol has important roles in the body in
membranes. It as also a precursor of steroid hormones and bile
acids and its immediate
metabolic precursor, 7dehydrocholesterol, is a
precursor of Vitamin D. The
pathway leading to cholesterol is

on Steroid and Lipid
Metabolism and Lipid
Movement in the Body HERE
and HERE
157
enzyme is regulated both by feedback inhibition (cholesterol

inhibits it) and by covalent modification (phosphorylation inhibits
The Pathway to Cholesterol
known as the isoprenoid pathway and branches of it lead to

other molecules including other fat-soluble vitamins.
From HMG-CoA, the enzyme HMG-CoA reductase catalyzes the
formation of mevalonate. The reaction requires NADPH and
results in release of coenzyme A and appears to be one of the
most important regulatory steps in the synthesis pathway. The
Steroid Hormone Synthesis

158
Mevalonate gets phosphorylated twice and
To Make a Cholesterol
then decarboxylated to yield the five carbon
To the tune of When Johnny Comes Marching Home
intermediate known as isopentenylpyrophosphate (IPP). IPP is readily
Some things that you can build with acetyl-CoAs

Are joined together partly thanks to thiolase
They come together 1-2-3
Six carbons known as H-M-G
And youre on your way
To make a cholesterol
converted to dimethylallylpyrophosphate
A single step links farnesyls but thats not all
The squalene rearranges to lanosterol
From that theres nineteen steps to go
Before the sterols apropos
Which you must recall
To make a cholesterol
The regulation of the schemes complex in ways

Inhibited by feedback of the RE-duc-tase
And statins mimic so they say
The look of HMG-CoA
So we sing their praise
And not make cholesterol
To synthesize a mevalonate in the cell

Requires reducing HMG-CoA, as well
The enzyme is a RE-ductase
Controlled in allosteric ways
When the cell's impelled
To make a cholesterol.
The mevalonate made in metabolic schemes

Gets decarboxylated down to isoprenes
Theyre linked together willy-nil
To build a PP-geranyl
In the cells routinesTo make a cholesterol
(DMAPP). These two five carbon

compounds, also called isoprenes, are the
building blocks for the synthesis of
cholesterol and related compounds. This
pathway is known as the isoprenoid
pathway. It proceeds in the direction of
cholesterol starting with the joining of IPP
and DMAPP to form geranylpyrophosphate. Geranyl-pyrophosphate
combines with another IPP to make
farnesyl-pyrophosphate, a 15-carbon
compound.
Two farnesyl-pyrophosphates join to

create the 30-carbon compound known as
squalene. Squalene, in a complicated

rearrangement involving reduction and
molecular oxygen forms a cyclic
it). The enzymes synthesis is also regulated transcriptionally.
intermediate known as lanosterol that resembles cholesterol.
When cholesterol levels fall, transcription of the gene increases.
Conversion of lanosterol to cholesterol is a lengthy process

involving 19 steps that occur in the endoplasmic reticulum.
159
Branching from cholesterol, one can form Vitamin D or the

steroid hormones, which include the progestagens, androgens,
estrogens, mineralocorticoids, and the glucocorticoids (pictured
on the previous page). The branch molecule for all of these is the
cholesterol metabolite (and progestagen) known as
pregnenalone. The progestagens are precursors of all of the
other classes.
The estrogens are derived from the androgens in an interesting
reaction that required formation of an aromatic ring. The enzyme
catalyzing this reaction is known as an aromatase and it is of
medical significance. The growth of some tumors is stimulated
by estrogens, so aromatase inhibitors are prescribed to prevent
the formation of estrogens and slow tumor growth.
It is worth noting that synthesis of other fat soluble vitamins and
chlorophyll also branches from the isoprenoid synthesis
Bile Salts
pathway at geranylpyrophosphate. Joining of two

geranylgeranylpyrophosphates occurs in plants and bacteria and
Converting the very non-polar cholesterol to a bile acid involves
leads to synthesis of lycopene, which, in turn is a precursor of
oxidation of the terminal carbon on the side chain o the rings.
beta-carotene, the final precursor of Vitamin A. Vitamins E and
Other alterations to increase the polarity of these compounds
K, as well as chlorophyll are all also synthesized from
include hydroxylation of the rings and linkage to other polar
geranylgeranylpyrophosphate.
compounds.
Bile Acid Metabolism
Common bile acids include cholic acid, chenodeoxycholic
Another pathway from cholesterol leads to the polar bile acids,

which are important for the solubilization of fat during digestion.
acid, glycocholic acid, taurocholic acid, and deoxycholic

acid. Another important fact about bile acids is that their
synthesis reduces the amount of cholesterol available and
160
somewhat unstable, chemically. It will decarboxylate

spontaneously to some extent to yield acetone.
Ketone bodies are made when the blood levels of glucose fall
very low. Ketone bodies can be converted to acetyl-CoA, which
can be used for ATP synthesis via the citric acid cycle. People
who are very hypoglycemic (including some diabetics) will
produce ketone bodies and these are often first detected by the
smell of acetone on their breath.
Acetone is of virtually no use for energy production since it is not
readily converted to acetyl-CoA. Consequently, cells convert
acetoacetate into beta-hydroxybutyrate, which is more chemically
stable. Though technically not a ketone, beta-hydroxybutyrate is
frequently referred to as a ketone body. Upon arrival at a target
cell, it can be oxidized back to acetoacetate with conversion to
acetyl-CoA. Both acetoacetate and beta-hydroxybutyrate can
Ketone Body Reactions
cross the blood-brain barrier and provide important energy for the
promotes uptake of LDLs by the liver. Normally bile acids are
brain when glucose is limiting.
recycled eciently resulting in limited reduction

of cholesterol levels. However, inhibitors of the
recycling promote reduction of cholesterol
levels.
See Kevins lecture on

Ketone Bodies HERE
Prostaglandin Synthesis
The pathway for making prostaglandins is an
extension of the fatty acid synthesis pathway (below).
Prostaglandins, molecules associated with localized
Ketone Body Metabolism
pain, are synthesized in cells from arachidonic acid (see previous
In ketone body synthesis, an acetyl-CoA is split o from HMG-
page) which has been cleaved from membrane lipids. The
CoA, yielding acetoacetate, a four carbon ketone body that is
enzyme catalyzing their synthesis is known as prostaglandin

161
Prostaglandins
To the tune of Oklahoma
Prossss-taglandins
The ei-co-sa-noids creating pain
Are the ones to blame - when you get inflamed
And ouch(!) - they hurt inside your brain
Prossss-taglandins
Every throb and ache gets magnified
If you hope to win, cyclo-oxygen's
Generation's got to be denied
The Vioxx has all been recalled
So go get yourself Tylenol-ed
And if you aaaaaaaaaaaaache
Blame PGH synthaaaaaaaaase!
We must complain that
You make the aches prostaglandins
Prostaglandin - D2, F1, G2, E2
Prostaglandin, it's you
enzyme is a strategy of non-steroidal pain relievers (also called

NSAIDs), such as aspirin or ibuprofen. Inhibition of the release
of arachidonic acid from membranes is the mechanism of action
of steroidal anti-inflammatories, which inhibit the
phospholipaseA2 (PLA2) that catalyzes the cleavage reaction.
Fatty
Acid
Oxidation
Breakdown of
fats yields fatty
acids and
glycerol.
Glycerol can be
readily
Prostaglandin Metabolism
converted to
DHAP for oxidation in glycolysis or synthesis into glucose in
gluconeogenesis. Fatty acids are broken down in two carbon
units of acetyl-CoA. To be oxidized, they must be transported
through the cytoplasm attached to coenzyme A and moved into

mitochondria. The latter step requires removal of the CoA and
attachment of the fatty acid to a

molecule of carnitine. The
carnitine complex is transported

on Fat, Fatty Acid, and
synthase, but is more commonly referred to as a
across the inner membrane of
Prostaglandin Metabolism
cyclooxygenase (or COX) enzyme. Inhibition of the action of this
the mitochondrion after which

162
ketone; and 4) thiolytic cleavage to release acetyl-CoA and a

fatty acid two carbons shorter than the starting one.
Unsaturated fatty acids complicate the picture a bit (see below),
primarily because they have cis bonds, for the most part, if they
are of biological origin and these must be converted to the
relevant trans intermediate made in step 1. Sometimes the bond
must be moved down the chain, as well, in order to be
positioned properly. Two enzymes (described below) handle all
the necessary isomerizations and moves necessary to oxidize all
of the unsaturated fatty acids.
Enzymes of Beta Oxidation

The reactions of fatty acid oxidation are notable in mirroring the
oxidations in the latter half of the citric acid cycle
Movement of Acyl-CoAs into the Mitochondrial Matrix
the fatty acid is reattached to coenzyme A in the mitochondrial

matrix.
dehydrogenation of succinate to make a trans double bond

(fumarate), hydration across the double bond to make L-malate
and oxidation of the hydroxyl to make a ketone (oxaloacetate).
Two of the enzymes of beta-oxidation are notable. The first is
acyl-CoA dehydrogenase, which catalyzes the initial
The process of fatty acid oxidation, called beta oxidation, is fairly
dehydrogenation and yields FADH2. It comes in three dierent
simple. The reactions all occur between carbons 2 and 3 (with #1
forms ones that work on long, medium, or short chain length
being the one linked to the CoA) and sequentially include the
fatty acids. The first of these is sequestered in the peroxisome of
following 1) dehydrogenation to create FADH2 and a fatty acyl
animals whereas the others are found in the mitochondria. Plants
group with a double bond in the trans configuration; 2) hydration
and yeast perform beta oxidation exclusively in the peroxisome.
across the double bond to put a hydroxyl group on carbon 3 in
The most interesting of the acyl-CoA dehydrogenases is the one
the L configuration; 3) oxidation of the hydroxyl group to make a
that works on medium length fatty acids. This one, which is the
163
one most commonly

deficient in animals, has
See Kevins lectures on fat,

Fatty Acid, and Prostaglandin
been linked to sudden
Metabolism HERE, HERE,
infant death syndrome.
and HERE
Reactions two and three in

beta oxidation are catalyzed by enoyl-CoA hydratase and
3-hydroxyacyl-CoA dehydrogenase, respectively. The latter
reaction yields an NADH. The final enzyme of beta
oxidation is thiolase and this enzyme is notable in not only
catalyzing the formation of acetyl-CoAs in beta oxidation,
but also catalyzing the joining of two acetyl-CoAs
(essentially the reversal of the last step of beta oxidation) to
form acetoacetyl-CoA essential for the pathways of
ketone body synthesis and cholesterol biosynthesis.
Oxidation of Odd Chain Fatty Acids

Though most fatty acids of biological origin have even
Beta Oxidation of Fatty Acids
In beta oxidation, it just occurred to me

The process all takes place tween carbons two and three
Some hydrogens are first removed to FADH2
Then water adds across the bond, the H to carbon two
Hydroxyl oxidations next, a ketone carbon three
Then thiolase catalysis dissects the last two Cs
The products of the path, of course, are acetyl-CoAs
Unless there were odd carbons, hence propionyl-CoA
164
Oxidation of Odd Chain Fatty Acids and Related Compounds
numbers of carbons, not all of them do. Oxidation of fatty acids

with odd numbers of carbons ultimately produces an intermediate
with three carbons called propionyl-CoA, which cannot be
oxidized further in the beta-oxidation pathway. Metabolism of
this intermediate is odd. Sequentially, the following steps occur
1) carboxylation to make D-methylmalonyl-CoA; 2) isomerization
to L-methylmalonyl-CoA; 3) rearrangement to form succinyl-CoA.
The last step of the process utilizes the enzyme methylmalonylCoA mutase, which uses the B12 coenzyme in its catalytic cycle.
Succinyl-CoA can then be metabolized in the citric acid cycle.

As noted above, oxidation of unsaturated fatty acids requires two
additional enzymes to the complement of enzymes for beta
oxidation. If the beta oxidation of the fatty acid produces an
intermediate with a cis bond between carbons three and four, cis-

165
3-Enoyl-CoA Isomerase will convert the bond to a trans bond
the reactions of the pathway leads to Refsums disease where
between carbons two and three and beta oxidation can proceed
accumulation of phytanic acid leads to neurological damage.
as normal.
On the other hand, if beta oxidation produces an intermediate
with a cis double bond between carbons four and five, the first
step of beta oxidation (dehydrogenation between carbons two
and three) occurs to produce an intermediate with a trans double
bond between carbons two and three and a cis double bond
between carbons four and five. The enzyme 2,4 dienoyl CoA
reductase reduces this intermediate (using NADPH) to one with a
single cis bond between carbons three and four. This
intermediate is then identical to the one acted on by cis-3Enoyl-CoA Isomerase above, which converts it into a regular beta
oxidation intermediate, as noted above.
Alpha Oxidation
Yet another consideration for oxidation of fatty acids is alpha

Synthesis of fatty acids occurs in the cytoplasm and endoplasmic
reticulum of the cell and is chemically similar to the betaoxidation process, but with a couple of key dierences. The first
of these occur in preparing substrates for the reactions that grow
the fatty acid. Transport of acetyl-CoA from the mitochondria
occurs when it begins to build up. Two molecules can play roles
in moving it to the cytoplasm citrate and acetylcarnitine.
Joining of oxaloacetate with acetyl-CoA in the mitochondrion
creates citrate which moves across the membrane, followed by
action of citrate lyase in the cytoplasm of the cell to release
acetyl-CoA and oxaloacetate. Additionally, when free acetyl-CoA
accumulates in the mitochondrion, it may combine with carnitine
and be transported out to the cytoplasm.
oxidation. This pathway is necessary for catabolism of fatty acids
Starting with two acetyl-CoA, one is converted to malonyl-CoA
that have branches in their chains. For example, breakdown of
by carboxylation catalyzed by the enzyme acetyl-CoA
chlorophylls phytol group yields phytanic acid, which undergoes
carboxylase (ACC), the only regulatory enzyme of fatty acid
hydroxylation and oxidation on carbon number two (in contrast to
synthesis (see below). Next, both molecules have their CoA
carbon three of beta oxidation), followed by decarboxylation and
portions replaced by a carrier protein known as ACP (acyl-carrier
production of a branched intermediate that can be further

oxidized by the beta oxidation pathway. Though alpha oxidation
is a relatively minor metabolic pathway, the inability to perform
protein) to form acetyl-ACP and malonyl-ACP. Joining of a fatty

acyl-ACP (in this case, acetyl-ACP) with malonyl-ACP splits out
166
intermediate of beta oxidation, the beta intermediate here is in

the D-configuration. Next, water is removed from carbons 2
and 3 of the hydroxyl intermediate to produce a trans doubled
bonded molecule. Last, the double bond is hydrogenated to
yield a saturated intermediate. The process cycles with the
addition of another malonyl-ACP to the growing chain until
ultimately an intermediate with 16 carbons is produced
(palmitoyl-CoA). At this point, the cytoplasmic synthesis
ceases.
For fatty acid synthesis, I must reverse the path

Of breaking fatty acids, though youll wonder bout the math
Each cycle of addition starts with carbons one two three
Yet products of reactions number carbons evenly
The answer is that CO2 plays peek-a-boo like games
By linking to an Ac-CoA then popping o again
Reactions are like oxidations cept theyre backwards here
Reduction, dehydration, then two hydrogens appear
the carboxyl that was added and creates the intermediate at the
upper right in the figure at left.
The product of the process is a 16 carbon chain

The bonds are saturated, no double ones remain
For them desaturases toil to put in links of cis
In animals to delta nine, but no more go past this
From this point forward, the chemical reactions resemble those of

beta oxidation reversed. First, the ketone is reduced to a
hydroxyl using NADPH. In contrast to the hydroxylated
And last theres making longer ones eicosanoidic fun

Theyre made by elongases in the e. reticulum
167
When Acids Are Synthesized

To the tune of When Johnny Comes Marching Home
The 16 carbon fatty acid, palmitate

Gets all the carbons that it needs from acetate
Which citric acid helps release
From mitochondri - matrices
Oh a shuttle's great
When acids are synthesized
Carboxylase takes substrate and it puts within
Dioxy carbon carried on a biotin
CoA's all gain a quick release
Replaced by larger ACPs
And it all begins
A malonate contributes to the growing chain
Two carbons seven times around again, again
For saturated acyl-ates
There's lots of N-A-DPH
That you must obtain
Palmitic acid made this way all gets released
Desaturases act to make omega-threes
The finished products big and small
Form esters with a glycerol
So you get obese
Enzymes of Fatty Acid Synthesis

Acetyl-CoA carboxylase, which catalyzes synthesis of malonylCoA, is the only regulated enzyme in fatty acid synthesis. Its
regulation involves both allosteric control and covalent
modification. The enzyme is known to be phosphorylated by
both AMP Kinase and Protein Kinase A. Dephosphorylation is
stimulated by phosphatases activated by insulin binding.
Dephosphorylation activates the enzyme and favors its assembly
into a long polymer, while phosphorylation reverses the process.
Citrate acts as an allosteric activator and may also favor
polymerization. Palmitoyl-CoA allosterically inactivates it.
In animals, six dierent catalytic activities necessary for the
remaining catalytic actions to fully make palmitoyl-CoA are
contained in a single complex called Fatty Acid Synthase.
These include transacylases for swapping CoA with ACP on
acetyl-CoA and malonyl-CoA; a synthase to catalyze addition of
the two carbon unit from the three carbon malonyl-ACP in the
first step of the elongation process; a reductase to reduce the
ketone; a dehydrase to catalyze removal of water, and a
reductase to reduce the trans double bond. In bacteria, these
activities are found on separate enzymes and are not part of a
complex.

Fatty Acid Elongation

Elongation to make fatty acids longer than 16 carbons occurs in
168
the endoplasmic reticulum and is catalyzed by
Metabolism of Fat
enzymes described as elongases. Mitochondria
Breakdown of fat in adipocytes requires
also can elongate fatty acids, but their starting
catalytic action of three enzymes, hormone
materials are generally shorter than 16 carbons
sensitive triacylglycerol lipase (called LIPE)
long. The mechanisms in both environments are
to remove the first fatty acid from the fat,
similar to those in the cytoplasm (a malonyl group
diglyceride lipase to remove the second one
is used to add two carbons, for example), but
and monoglyceride lipase to remove the third.
CoA is attached to the intermediates, not ACP.
Of these, only LIPE is regulated and it appears
Further, whereas cytoplasmic synthesis employs
to be the rate limiting reaction. Synthesis of fat
an enzyme complex called fatty acid synthase,
starting with glycerol-3-phosphate requires
the enzymes in these organelles are separable
action of acyl transferase enzymes, such as
and not part of a complex.
glycerol-3-phosphate acyl transferase, which
Desaturation of Fatty Acids
catalyze addition of fatty acids to the glycerol

backbone.
Fatty acids are synthesized in the saturated form

and desaturation occurs later. Enzymes called
Interestingly, there appear to be few controls
desaturases catalyze the formation of cis double
of the metabolism of fatty acids. The primary
bonds in mature fatty acids. These enzymes are
control of their oxidation is availability. One
found in the endoplasmic reticulum. Animals are
way to control that is by control of the
limited in the desaturated fatty acids they can
breakdown of fat. This process, which can be
make, due to an inability to catalyze reactions
stimulated by the epinephrine kinase cascade,
beyond carbons 9 and 10. Thus, humans can
is controlled through LIPE, found in adipocytes
make oleic acid, but cannot synthesis linoleic
(fat-containing cells). Breakdown of fat in
acid or linolenic acid. Consequently, these two
apidocytes requires action of three enzymes,
must be provided in the diet and are referred to as
each hydrolyzing one fatty acid from the
essential fatty acids.
Synthesis of Fat
glycerol backbone. As noted earlier, only

169
HSTL, which catalyzes the first hydrolysis, is regulated.

Synthesis of fat requires glycerol-3-phosphate (or DHAP)
and three fatty acids. In the first reaction, glycerol-3phosphate is esterified at position 1 with a fatty acid,
followed by a duplicate reaction at position 2 to make
phosphatidic acid. This molecule, which is an intermediate
in the synthesis of both fats and phosphoglycerides, gets
dephosphorylated to form diacylglycerol before the third
esterification to make a fat.
Glycerophospholipid Metabolism
Phosphatidic acid, as noted above, is an important
intermediate in the metabolism of glycerophospholipids.
These compounds, which are important membrane
constituents, can be synthesized in several ways.
Connections to Other Pathways

There are several connections between metabolism of fats
and fatty acids to other metabolic pathways. As noted,
phosphatidic acid is an intermediate in the synthesis of
triacylglycerols, as well as of other lipids, including
phosphoglycerides. Diacylglycerol (DAG), which is an
intermediate in fat synthesis, also acts as a messenger in
some signaling systems. Fatty acids twenty carbons long
Activation of Triacylglycerol Lipase and the Breakdown of Fat
based on arachidonic acid (also called eicosanoids) are

precursors of the classes of molecules known as
170
leukotrienes and prostaglandins. The latter, in turn, are

precursors of the class of molecules known as thromboxanes.
The ultimate products of beta oxidation are acetyl-CoA molecules
and these can be assembled by the enzyme thiolase to make
acetoacetyl-CoA, which is a precursor of both ketone bodies and
the isoprenoids, a broad category of compounds that include
steroid hormones, cholesterol, bile acids, and the fat soluble
vitamins, among others.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
171
Chapter 7
Metabolism II
In this second section of metabolism, we cover

metabolic pathways that do not have a strong
emphasis on oxidation/reduction.
Metabolism II
Carbohydrate Storage and Breakdown
In the last chapter, we focused on metabolic pathways that played important
Glycogen Breakdown
oxidative/reductive roles relative to cellular energy. In this chapter, the pathways
Regulation of Glycogen Metabolism
that we cover have lesser roles from an energy perspective, but important roles,
GPa/GPb Allosteric Regulation
nonetheless, in catabolism and anabolism of building blocks of proteins and
GPa/GPb Covalent Conversion
nucleic acids, nitrogen balance, and sugar balance. In a sense, these might be
Turning O Glycogen Breakdown
thought of as the kitchen sink pathways, but it should be noted that all cellular
Glycogen Synthesis
Regulation of Glycogen Synthesis
Maintaining Blood Glucose Levels
pathways are important.
Carbohydrate Storage/Breakdown
Pentose Phosphate Pathway
Carbohydrates are important cellular energy sources. They provide energy quickly
Calvin Cycle
through glycolysis and passing of intermediates to pathways, such as the citric
C4 Plants
acid cycle, amino acid metabolism (indirectly), and the pentose phosphate
Urea Cycle
pathway. It is important, therefore, to understand how these important molecules
Nitrogen Fixation
are made.
Amino Acid Metabolism

Amino Acid Catabolism
Nucleotide Metabolism
Pyrimidine de novo Biosynthesis
Purine de novo Biosynthesis
Deoxyribonucleotide de novo Biosynthesis
Plants are notable in storing glucose for energy in the form of amylose and
amylopectin (see HERE) and for structural integrity in the form of cellulose (see
HERE). These structures dier in that cellulose contains glucoses solely joined by
beta-1,4 bonds, whereas amylose has only alpha1,4 bonds and amylopectin has
alpha 1,4 and alpha 1,6 bonds.
173
Animals store glucose primary in liver and muscle in the form of a
Breakdown of glycogen involves 1) release of glucose-1-
compound related to amylopectin known as glycogen. The
phosphate (G1P), 2) rearranging the remaining glycogen (as

necessary) to permit continued breakdown, and 3) conversion of
G1P to G6P for further metabolism. G6P can be 1) broken down
in glycolysis, 2) converted to glucose by gluconeogenesis, and 3)
oxidized in the pentose phosphate pathway.
Just as in gluconeogenesis, the cell has a separate mechanism
for glycogen synthesis that is distinct from glycogen breakdown.
As noted previously, this allows the cell to separately control the
reactions, avoiding futile cycles, and enabling a process to occur
eciently (synthesis of glycogen) that would not occur if it were
The Repeating Structure of Cellulose
structural dierences between glycogen and amylopectin are

solely due to the frequency of the alpha 1,6 branches of glucoses.
In glycogen they occur about every 10 residues instead of every
simply the reversal of glycogen breakdown.

Synthesis of glycogen starts with G1P, which is converted to an
'activated' intermediate, UDP-glucose. This activated
30-50, as in amylopectin.
Glycogen provides an additional source of glucose besides that
produced via gluconeogenesis. Because glycogen contains so
many glucoses, it acts like a battery backup for the body,
providing a quick source of glucose when needed and providing a
place to store excess glucose when glucose concentrations in the
blood rise. The branching of glycogen is an important feature of
the molecule metabolically as well. Since glycogen is broken
down from the "ends" of the molecule, more branches translate
to more ends, and more glucose that can be released at once.
The Repeating Unit of Glycogen
intermediate is what 'adds' the glucose to the growing glycogen

174
chain in a reaction catalyzed by the enzyme known as

glycogen synthase. Once the glucose is added to
glycogen, the glycogen molecule may need to have
branches inserted in it by the enzyme known as
branching enzyme.
Glycogen Breakdown
Glycogen phosphorylase (sometimes simply called
Phosphorolysis of Glycogen
phosphorylase) catalyzes breakdown of glycogen into

Glucose-1-Phosphate (G1P). The reaction, (see above
right) that produces G1P from glycogen is a phosphorolysis, not a
hydrolysis reaction. The distinction is that hydrolysis
enzyme also catalyzes the hydrolysis of the remaining glucose at

the 1,6 branch point. Thus, the breakdown products from
glycogen are G1P and glucose (mostly G1P,
reactions use water to cleave bigger molecules into

smaller ones, but phosphorolysis reactions use
phosphate instead for the same purpose. Note that
on Glycogen Metabolism
the phosphate is just that - it does NOT come from
ATP. Since ATP is not used to put phosphate on

G1P, the reaction saves the cell energy.
Glycogen phosphorylase will only act on non-reducing ends of a
glycogen chain that are at least 5 glucoses away from a branch
point. A second enzyme, Glycogen Debranching Enzyme (GDE),
is therefore needed to convert alpha(1-6) branches to alpha(1-4)
branches. GDE acts on glycogen branches that have reached
their limit of hydrolysis with glycogen phosphorylase. GDE acts to
transfer a trisaccharide from a 1,6 branch onto an adjacent 1,4
branch, leaving a single glucose at the 1,6 branch. Note that the
however). Glucose can, of course, be converted to

Glucose-6-Phosphate (G6P) as the first step in
glycolysis by either hexokinase or glucokinase.
G1P can be converted to G6P by action of an
enzyme called phosphoglucomutase. This reaction
is readily reversible, allowing G6P and G1P to be interconverted

as the concentration of one or the other increases. This is
important, because phosphoglucomutase is needed to form G1P
for glycogen biosynthesis.
Regulation of Glycogen Metabolism

Regulation of glycogen metabolism is complex, occurring both
allosterically and via hormone-receptor controlled events that
result in protein phosphorylation or dephosphorylation. In order
to avoid a futile cycle of glycogen synthesis and breakdown
175
simultaneously, cells have evolved an elaborate set of controls
phosphate (GPb here). GPb is converted to GPa by
that ensure only one pathway is primarily active at a time.
phosphorylation by an enzyme known as phosphorylase kinase.
Regulation of glycogen metabolism is managed by the enzymes

glycogen phosphorylase and glycogen synthase. Glycogen
GPa and GPb can each exist in an 'R' state and a 'T' state. For
both GPa and GPb, the R state is the more active form of the
enzyme. GPa's negative
phosphorylase is
allosteric eector
regulated by both
(glucose) is usually not
allosteric factors (ATP,
abundant in cells, so
G6P, AMP, and glucose)
GPa does not flip into
and by covalent
the T state often. There
modification
is no positive allosteric
(phosphorylation/
eector of GPa, so when
dephosphorylation). Its
glucose is absent, GPa
regulation is consistent
automatically flips into
with the energy needs of
the R (more active) state.
the cell. High energy

substrates (ATP, G6P,
GPb can convert from
glucose) allosterically
the T state to the GPb R
inhibit GP, while low
state by binding AMP.
energy substrates (AMP,
Unless a cell is low in
others) allosterically
energy, AMP
activate it.
Regulation of Glycogen Phosphorylase
concentration is low.
Thus GPb is not
GPa/GPb
Allosteric Regulation
very often. On the other hand, ATP and/or G6P are usually
Glycogen phosphorylase exists in two dierent covalent forms
present at high enough concentration in cells that GPb is readily
one form with phosphate (called GPa here) and one form lacking
flipped into the T state.
converted to the R state
176
receptors, also known as G-protein coupled receptors. These are
GPa/GPb Covalent Conversion
discussed in greater detail in Chapter 8.
Because the relative amounts of
Common ligands for these receptors
GPa and GPb largely govern the
include epinephrine (binds beta-
overall process of glycogen
adrenergic receptor) and glucagon
breakdown, it is important to
(binds glucagon receptor). Epinephrine
understand the controls on the
exerts it greatest eects on muscle and
enzymes that interconvert GPa
glucagon works preferentially on the
and GPb. This is accomplished by
liver.
the enzyme Phosphorylase Kinase,

which transfers phosphates from 2
Turning O Glycogen
Breakdown
ATPs to GPb to form GPa.

Phosphorylase kinase has two
Turning OFF signals is as important, if
covalent forms phosphorylated
not more so, than turning them ON. The
(active) and dephosphorylated
steps in the glycogen breakdown
(inactive). It is phosphorylated by
regulatory pathway can be reversed at
the enzyme Protein Kinase A
several levels. First, the ligand can leave
(PKA). Another way to activate the
the receptor. Second, the G-proteins
enzyme is with calcium.
have an inherent GTPase activity that
Phosphorylase kinase is
Hormone Signaling Through the Kinase Cascade to
dephosphorylated by the same
Stimulate Glycogen Breakdown
enzyme, phosphoprotein
phosphatase, that removes
serves to turn them o over time. Third,

cells have phosphodiesterase (inhibited
by caeine) for breaking down cAMP.
phosphate from GPa.
Fourth, an enzyme known as

phosphoprotein phosphatase can
PKA is activated by cAMP, which is, in turn produced by
remove phosphates from phosphorylase kinase (inactivating it)
adenylate cyclase after activation by a G-protein. G-proteins are
AND from GPa, converting it to the much less active GPb.
activated ultimately by binding of ligands to specific 7-TM

177
synthase recognition is catalyzed by a protein called glycogenin,
Glycogen Synthesis
The anabolic pathway contrasting with glycogen breakdown is
that of glycogen synthesis. Just as cells reciprocally regulate
glycolysis and gluconeogenesis to prevent a futile cycle, so too
do cells use reciprocal schemes to regulate glycogen breakdown
and synthesis. Let us first consider the steps in glycogen
which attaches to the first glucose and catalyzes linkage of the

first eight glucoses by alpha(1,4) bonds. 3) The characteristic
alpha(1,6) branches of glycogen are the products of an enzyme
known as Branching Enzyme. Branching Enzyme breaks
alpha(1,4) chains and carries the broken chain to the carbon #6
and forms an alpha(1,6) linkage.
synthesis. 1) Glycogen synthesis from glucose involves

phosphorylation to form G6P, and isomerization to form G1P
Regulation of Glycogen Synthesis
(using phosphoglucomutase common to

glycogen breakdown). G1P is reacted with
Interactive 7.1
The regulation of glycogen biosynthesis is

reciprocal to that of glycogen breakdown. It
UTP to form UDP-glucose in a reaction
also has a cascading covalent modification
catalyzed by UDP-glucose
system similar to the glycogen breakdown
pyrophosphorylase. Glycogen synthase
system described above. In fact, part of the
catalyzes synthesis of glycogen by joining
system is identical to glycogen breakdown.
carbon #1 of the UDPG-derived glucose onto
Epinephrine or glucagon signaling can
the carbon #4 of the non-reducing end of a
stimulate adenylate cyclase to make cAMP,
glycogen chain. to form the familiar alpha(1,4)
The 3D Structure of Insulin
glycogen links. Another product of the
which activates PKA, which activates

phosphorylase kinase.
reaction is UDP.
In glycogen breakdown, phosphorylase kinase phosphorylates
It is also worth noting in passing that glycogen synthase will only
GPb to the more active form, GPa. In glycogen synthesis, protein
add glucose units from UDPG onto a preexisting glycogen chain
kinase A phosphorylates the active form of glycogen synthase
that has at least four glucose
(GSa), and converts it into the usually inactive b form (called
residues. Linkage of the first few
GSb). Note the conventions for glycogen synthase and glycogen
on Glycogen Metabolism
glucose units to form the minimal
phosphorylase. For both enzymes, the more active forms are
"primer" needed for glycogen
called the 'a' forms (GPa and GSa) and the less active forms are
178
is activated when insulin binds to a receptor in the cell

membrane. It causes PP to be activated, stimulating
dephosphorylation, and thus activating glycogen synthesis and
inhibiting glycogen breakdown. Again, there is reciprocal
regulation of glycogen synthesis and degradation.
Maintaining Blood Glucose Levels

After a meal, blood glucose levels rise and insulin is released. It
simultaneously stimulates uptake of glucose by cells and
incorporation of it into glycogen by activation of glycogen
synthase and inactivation of glycogen phosphorylase. When
blood glucose levels fall, GPa gets activated (stimulating
glycogen breakdown to raise blood glucose) and GSb is formed
Glycogen Synthase
called the 'b' forms (GPb and GSb). The major dierence,
however, is that GPa has a phosphate, but GSa does not and
GPb has no phosphate, but GSb does. Thus phosphorylation and
dephosphorylation have opposite eects on the enzymes of
glycogen metabolism. This is the hallmark of reciprocal
regulation. It is of note that the less active glycogen synthase
(stopping glycogen synthesis).
Pentose Phosphate Pathway

The Pentose Phosphate Pathway (PPP) is one that many students
are confused by. Perhaps the reason for this is that it doesnt
really have a single direction in which it proceeds, as will be
apparent below.
form, GSb, can be activated by G6P. Recall that G6P had the
Portions of the PPP are similar to the Calvin Cycle of plants, also
exactly opposite eect on GPb.
known as the dark reactions of photosynthesis. We discuss
Glycogen synthase, glycogen phosphorylase (and phosphorylase

kinase) can be dephosphorylated by several enzymes called
phosphatases. One of these is called Protein Phosphatase and it
these reactions separately in the next section. The primary

functions of the PPP are to produce NADPH (for use in anabolic
reductions), ribose-5-phosphate (for making nucleotides), and
erythrose-4-phosphate (for making aromatic amino acids). Three
179
molecular intermediates of glycolysis can funnel into PPP

(or be used as usual in glycolysis). They include G6P,
fructose-6-phosphate (in two places), and
glyceraldehyde-3-phosphate (also in two places).
A starting point for the pathway (though there are other
entry points) is the oxidative phase. It includes two
reactions generating NADPH. In the first of these,
oxidation of glucose-6-phosphate (catalyzed by
glucose-6-phosphate dehydrogenase), produces
NADPH and 6-phosphogluconolactone. 6phosphogluconolactone spontaneously gains water and
loses a proton to become 6-phosphogluconate.
Oxidation of this produces ribulose-5-phosphate and
another NADPH and releases CO2. The remaining steps of
the pathway are known as the non-oxidative phase and
involve interconversion of sugar phosphates.
For example, ribulose-5-phosphate is converted to
ribose-5-phosphate (R5P) by the enzyme ribulose-5phosphate isomerase. Alternatively, ribulose-5phosphate can be converted to xylulose-5-phosphate
(Xu5P). R5P and Xu5P (10 carbons total) can be combined
and rearranged by transketolase to produce intermediates
with 3 and 7 carbons (glyceraldehyde-3-phosphate and
sedoheptulose-7-phosphate, respectively). These last
two molecules can, in turn be rearranged by transaldolase
The Pentose Phosphate Pathway

180
into 6 and 4 carbon sugars

(fructose-6-phosphate and
erythrose-4-phosphate,
respectively). Further, the
erythrose-4-phosphate can swap
parts with Xu5P to create
glyceraldehyde-3-phosphate and
fructose-6-phosphate.
It is important to recognize that
the PPP pathway is not a topdown pathway, with all the
intermediates derived from a
starting G6P. All of the reactions
are reversible, so that, for
example, fructose-6-phosphate
and glyceraldehyde-3-phosphate
from glycolysis can reverse the
last reaction of the previous
paragraph to provide a means of
synthesizing ribose-5-phosphate
non-oxidatively. The pathway
also provides a mechanism to
cells for metabolizing sugars,
such as Xu5P and ribulose-5phosphate. In the bottom line of
the pathway, the direction the
I need erythrose phosphate

And dont know what to do
My cells are full of G-6-P
Plus NADP too
But I just hit upon a plan
As simple as can be
Ill run reactions through the path
Thats known as PPP
In just two oxidations
Theres ribulose-5P
Which morphs to other pentoses
Each one attached to P
The next step it is simple
Deserving of some praise
The pentose carbons mix and match
Thanks to transketolase
Glyceraldehydes a product
Sedoheptulose is too
Each with a trailing phosphate
But we are not quite through
Now three plus seven is the same
As adding six and four
By swapping carbons back and forth
Theres erythrose-P and more
At last Ive got the thing I need
From carbons trading places
Im happy that my cells are full
Of these transaldolases
pathway goes and the intermediates it

produces are determined by the needs
of, and intermediates available to, the
cell.
As noted above, the pathway connects
in three places with glycolysis. In nonplant cells, the PPP pathway occurs in
the cytoplasm (along with glycolysis),
so considerable intermingling of
intermediates can and does occur.
Erythrose-4-phosphate is an important
precursor of aromatic amino acids and
ribose-5-phosphate is an essential
precursor for making nucleotides.
Calvin Cycle
The Calvin Cycle (next page) occurs
exclusively in photosynthetic
organisms and is the part of
photosynthesis referred to as the Dark
Cycle. It is in this part of the process
that carbon dioxide is taken from the
atmosphere and ultimately built into
glucose (or other sugars). Though
reduction of carbon dioxide to glucose
ultimately requires electrons from
181
twelve molecules of NADPH (and 18

ATPs), it is a bit confusing because one
reduction occurs 12 times (1,3 BPG to
G3P) to achieve the reduction necessary
to make one glucose.
One of the reasons students find the
pathway a bit confusing is because the
carbon dioxides are absorbed one at a
time into six dierent molecules of
ribulose-1,5-bisphosphate (Ru1,5BP). At
no point are the six carbons ever together
in the same molecule to make a single
glucose. Instead, six molecules of
Ru1,5BP (30 carbons) gain six more
carbons via carbon dioxide and then split
into 12 molecules of 3-phosphoglycerate
(36 carbons). The gain of six carbons
from Wikipedia
allows two three carbon molecules to be

produced in excess for each turn of the
cycle. These two molecules molecules are
then converted into glucose using the
enzymes of gluconeogenesis.
Like the citric acid cycle, the Calvin Cycle
doesnt really have a starting or ending
The Calvin Cycle
point, but can we think of the first reaction

182
as the fixation of carbon dioxide to

Ru1,5BP. This reaction is catalyzed
Photosynthesis is Divine
by the enzyme known as
To the tune of Scarborough Fair
ribulose-1,5bisphosphate
Photosynthesis is divine
Fixing carbon using sunshine
It's thanks to plants that we've got a prayer
They pull CO2 from the air
Carbon's fixed onto a substrate

Ribulose-1,5-bisphosphate
Rubisco acts in-e-fficient-ly
Splitting it into 3PGs
carboxylase (RUBISCO). The
Reaping energy from the sun

It's efficient second to none
You grab the photons almost at will
Protoporphyrin chlorophyll
If the enzyme grabs an O2

It makes glycolate, it is true
The Calvin Cycle works in a wheel
Giving plants a sugary meal
phosphoglycerate. As noted, if one
Light reactions of System II

Split up water, making O2
Electrons pass through schemes labeled 'Z'
Pumping protons gradiently
So photosynthesis is divine
'Cause it happens all of the time
From dawn to dusk and times in between
Solar panels truly are green
the cycle can be shunted o as two
resulting six carbon intermediate is

unstable and each Ru1,5BP is
rapidly converted to 3starts with 6 molecules of Ru1,5BP
and makes 12 molecules of 3-PG,
the extra 6 carbons that are a part of
three-carbon molecules of
glyceraldehyde-3-phosphate (GA3P)
to gluconeogenesis, leaving behind
10 molecules of GA3P to be
ATP's made due to a shift

Of the protons spinning quite swift
An enzyme turbine, cellular maze
You know as A-T-P synthase
reconverted into 6 molecules of

Ru1,5BP. That part of the pathway
requires multiple steps, but only
utilizes two enzymes unique to
plants -

lecture on Photosynthesis
Recording by David Simmons
HERE

183
sedoheptulose-1,7bisphosphatase
these plants, carbon dioxide is captured in
and phosphoribulokinase. RUBISCO
special mesophyll cells first by
is the third enzyme of the pathway that
phosphoenolpyruvate (PEP) to make
is unique to plants. All of the other
oxaloacetate. The oxaloacetate is converted
enzymes of the pathway are common
to malate and transported into bundle sheath
to plants and animals and include
cells where the carbon dioxide is released
some found in the pentose phosphate
and it is captured by ribulose-1,5-
pathway and gluconeogenesis.
bisphosphate, as in C3 plants and the Calvin

Cycle proceeds from there. The advantage
C4 Plants
of this scheme is that it allows concentration

of carbon dioxide while minimizing loss of
The Calvin Cycle is the means by
water and photorespiration.
which plants assimilate carbon dioxide

from the atmosphere, ultimately into
glucose. Plants use two general
Urea Cycle
strategies for doing so. The first is
Yet another cyclic pathway important in cells
employed by plants called C3 plants
is the urea cycle (next page). With reactions
(most plants) and it simply involves the
spanning the cytoplasm and the
pathway described above. Another
mitochondria, the urea cycle occurs mostly
class of plants, called C4 plants
in the liver and kidney. The cycle plays an
employ a novel strategy for
important role in nitrogen balance in cells
concentrating the CO2 prior to
and is found in organisms that produce urea
assimilation. C4 plants are generally
as a way to excrete excess amines.
found in hot, dry environments where
The cycle scavenges free ammonia (as
conditions favor the wasteful
ammonium ion) which is toxic if it
photorespiration reactions of
RUBISCO, as well as loss of water. In
accumulates. The capture reaction also

Assimilation of Carbon Dioxide in C4 Plants
requires ATP, and bicarbonate, and the

184
product is carbamoyl phosphate. This molecule is combined

with the non-protein amino acid known as ornithine to make
another non-protein amino acid known as citrulline. Addition of
aspartate to citrulline creates argninosuccinate, which splits o
a fumarate, creating arginine (a source of arginine). If arginine is
not needed, it can be hydrolyzed to yield urea (excreted) and
ornithine, thus completing the cycle.
The first two reactions described here occur in the mitochondrion
and the remaining ones occur in the cytoplasm. Molecules of the
urea cycle intersecting other pathways include fumarate (citric
acid cycle), aspartate (amino acid metabolism), arginine (amino
acid metabolism), and ammonia (amino acid metabolism).
Nitrogen Fixation
The process of nitrogen fixation is important for life on earth,
because atmospheric nitrogen is ultimately the source of amines
in proteins and DNA. The enzyme playing an important role in
this process is called nitrogenase and it is found in certain types
of anaerobic bacteria called diazotrophs. Symbiotic relationships
between some plants (legumes, for example) and the nitrogenfixing bacteria provide the plants with access to reduced nitrogen.
The overall reduction reaction catalyzed by nitrogenase is
N2 + 6H+ + 6e 2NH3
The Urea Cycle
185
In these reactions, the hydrolysis of 16 ATP is required.
They Call The Stuff Urea

To the tune of They Call The Wind Mariah
The ammonia can be assimilated into glutamate and

other molecules. Enzymes performing nitrogenase
catalysis are very susceptible to oxygen and must be
Get ATPs, bicarbonate,

Ammonia catalyzing
To make carba-mo-yl phosphate
And then start the synthesizing
Urea!
Urea!
Youve just made some urea!
The body handles many things
Requiring its attention
Like balancing aminos for
Uremia prevention
When joined up with an ornithine

In THE mi-TOE-chon-DREE-a
It turns into a citrulline
When cycling to urea
So if there's excess nitrogen

It is a good idea
To rid yourself of surplus by
Producing some urea
Urea!
Urea!
They call the stuff urea!
Urea!
Urea!
Go out and take a pee, yeah!
On exit to the cytosol

There's bonding aspartat-ic
The argininosuccinate
Is produced in this schematic
Urea!
Urea!
Have yourself a pee, yeah!
Bid farewell to a fumarate

Amino panacea
Arises when the arginine
Gets lysed to form urea
kept free of it. It is for this reason that most nitrogenfixing bacteria are anaerobic. Movement of amines
through biological systems occurs largely by the process
of transmination, discussed below in amino acid
metabolism.
Amino Acid Metabolism

The pathways for the
synthesis and
degradation of amino
on Nitrogen Metabolism
acids used in proteins
HERE and HERE
are the most varied

among the reactions synthesizing biological building
blocks. We start with some terms. First, not all
organisms can synthesize all the amino acids they need.
Amino acids that an organism cannot synthesize (and
therefore must have in their diets) are called essential
amino acids. The remaining amino acids that the body
can synthesize are called non-essential.

Amino acids are also divided according to the pathways
involved in their degradation. There are three general

categories. Ones that yield intermediates in the glycolysis
186
pathway are called glucogenic

and those that yield intermediates
Four Families of Synthesis of Essential Amino Acids
can be synthesized from citric

acid cycle intermediates. For
of acetyl-CoA or acetoacetate are
1. Aspartate lys, met, thr proceed through aspartyl-beta-
example, synthesis of the non-
called ketogenic. Those that
phosphate (catalyzed by aspartokinase)
essential amino acids occurs as
involve both are called glucogenic

and ketogenic.
An important general
consideration in amino acid
metabolism is that of
transamination. In this process,
an exchange of amine and oxygen
2. Pyruvate leu, ile, val proceed through hydroxyethyl-TPP

intermediate
follows: aspartic acid can be

made by transamination of
oxaloacetate. Glutamate comes
3. Aromatic phe, tyr, trp precursors are PEP and erythrose-4- from transamination of alphaketoglutarate. Pyruvate, as noted,
phosphate
is a precursor of alanine (via
4. Histidine from 5-phosphoribosyl-pyrophosphate (from
erythrose-4-phosphate), histidine (from ribose-5-phosphate),
between an amino acid and an

alpha-ketoacid occurs (see below)
Alpha-ketoacid + amino acid <=> amino acid + alpha-ketoacid
transamination). Amino acids that

can be made from glutamate
include glutamine (by addition of
an additional ammonium ion), proline, and arginine, Asparagine is

made from aspartate by addition of ammonium ion also. Serine is
formed from 3-phosphoglycerate and is itself the precursor of
An example reaction follows

Pyruvate + Aspartic acid <=> Alanine + Oxaloacetate
This reaction is catalyzed by an enzyme known as a
both glycine and cysteine. Cysteine and serine are also made
from methionine. Tyrosine is made by hydroxylation of
phenylalanine.
transaminase. Amino acids, such as glutamate, can also gain
Amino Acid Catabolism
nitrogen directly from ammonium ion, as shown below
Breakdown of glutamine by glutaminase is a source of
Alpha-ketoglutarate + NH4+ <=> Glutamate
ammonium ion in the cell. The other product is glutamate.

Glutamate, of course, can be converted by a transamination
This reaction can occur, for example, in nitrifying bacteria, and in
reaction to alpha-ketoglutarate, which can be oxidized in the citric
places where ammonia waste is produced. Many amino acids
acid cycle.
187
Asparagine can similarly be
and glycine. The other
broken to ammonium and
leads to pyruvate.
aspartate by asparaginase
Cysteine can be
and aspartate can be
broken down in
converted by transamination
several ways. The
to oxaloacetate for oxidation
simplest occurs in the
in the citric acid cycle.
liver, where a
Alanine is converted to
desulfurase can act on
pyruvate in a transamination
it to yield hydrogen
reaction, making it
sulfide and pyruvate.
glucogenic.
Methionine can be
Arginine is hydrolzyed in the
converted to cysteine
urea cycle to yield urea and
for further metabolism.
ornithine.
It can be converted to
Proline is catabolized to
Glucogenic and Ketogenic Amino Acids
glutamate in a reversal of its

synthesis pathway.
Serine donates a carbon to form a folate and the other product of
the reaction is glycine, which is itself oxidized to carbon dioxide
and ammonia. Glycine can also be converted back to serine,
which can also be converted back to 3-phosphoglycerate or
pyruvate.
Threonine can be broken down in three pathways, though only
two are relevant for humans. One pathway leads to acetyl-CoA
succinyl-CoA for
oxidation in the citric
acid cycle. It can also
be converted to S-Adenosyl-Methionine (SAM), a carbon donor.

Isoleucine and valine can also be converted to succinyl-CoA after
conversion first to propionyl-CoA. Since conversion of propionylCoA to succinyl-CoA requires vitamin B12, catabolism of these
amino acids also requires the vitamin.
Phenylalanine is converted during catabolism to tyrosine, which is
degraded ultimately to fumarate and acetoacetate. Thus, both of
188
these amino acids are
In summary, the following are
glucogenic and ketogenic.
metabolized to pyruvate alanine,
Tyrosine can also be
cysteine, glycine, serine, and
converted to dopamine,
threonine
norepinephrine, and
Oxaloacetate is produced from
epinephrine.
aspartate and asparagine
Leucine and lysine can be
Succinyl-CoA is produced from
catabolized to
isoleucine, valine, and methionine
acetoacetate and acetylCoA. Lysine is also an
Alpha-ketoglutarate is produced from
important precursor of
arginine, glutamate, glutamine,
carnitine.
histidine and proline.
Histidine can be
Phenylalanine and tyrosine are broken
catabolized by bacteria in
down to fumarate and acetoacetate
intestines to histamine,
Leucine and lysine yield acetoacetate
which causes construction
and acetyl-CoA.
or dilation of various blood

vessels when in excess.
Tryptophan leads to alanine,

acetoacetate and acetyl-CoA
Tryptophans catabolism is
complex, but can proceed
through alanine,
acetoacetate and acetylCoA
Last, amino acids, besides being

Conversion of L-tryptophan into Serotonin, Melatonin, and Niacin.
from Wikipedia
incorporated into proteins, serve as

precursors of important compounds,
including serotonin (from tryptophan),
porphyrin heme (from glycine), nitric oxide (from arginine), and

189
nucleotides (from aspartate, glycine,

and glutamine).
Arguably, the most interesting
metabolic pathways from the
perspective of regulation are those
leading to the synthesis of
nucleotides. We shall consider
ribonucleotide synthesis from from
scratch (de novo synthesis).
Deoxyribonucleotide synthesis from
ribonucleotides will be considered
separately.
Synthesis of ribonucleotides by the
de novo method occurs in two
pathways one for purines and one
for pyrimidines. What is notable
about both of these pathways is that
nucleotides are built from very simple
building blocks.
Pyrimidine de novo
Biosynthesis
Starting materials for pyrimidine biosynthesis (shown in the figure)
include bicarbonate, amine from glutamine, and phosphate from
De novo Synthesis of Pyrimidine Ribonucleotides
ATP to make carbamoyl-phosphate (similar to the reaction of the

urea cycle). Joining of carbamoyl phosphate to aspartic acid
190
See Kevins Nucleotide

Metabolism lectures on
YouTube HERE and HERE
(forming carbamoyl aspartate) is

catalyzed by the most important
regulatory enzyme of the cycle,
aspartate transcarbamoylase
(also called aspartate carbamoyltransferase or ATCase).

ATCase is regulated by three compounds. One of these
Purine de novo Biosynthesis

Synthesis of purine nucleotides diers fundamentally from that of
pyrimidine nucleotides in that the bases are built on the ribose
ring. The starting material is ribose-5-phosphate, which is
phosphorylated by PRPP synthetase to PRPP using two
phosphates from ATP. PRPP amidotransferase catalyzes the
(aspartate) is a substrate and it activates the enzyme by binding

to the catalytic site and favoring the enzymes R state. The
other two regulators bind to regulatory subunits of the enzyme
and either inhibit (CTP) or activate (ATP) the enzyme.
The reaction product, carbamoyl aspartate, is transformed in
two reactions to orotic acid, which is, in turn combined with
phosphoribosylpyrophosphate (PRPP). The product of that
reaction, orotidyl monophosphate (OMP) is decarboxylated to
form the first pyrimidine nucleotide, UMP. Conversion of UMP
to UDP is catalyzed by nucleoside monophosphate kinases
(NMPs) and UDP is converted to UTP by nucleoside
diphosphokinase (NDPK).
UDP (like all of the nucleoside diphosphates) is a branch point
to deoxyribonucleoside diphosphates, catalyzed by
ribonucleotide reductases, which are discussed later. UTP is
converted to CTP by CTP synthase. This enzyme, which uses
an amino group from glutamine for the reaction, serves to
balance the relative amounts of CTP and UTP, thanks to
inhibition by excess CTP.
De novo Synthesis of Purine Nucleotides

191
transfer of an amine group to PRPP, replacing the
but not stopped, thus allowing the reactions leading to IMP to
pyrophosphate on carbon 1. Thus begins the synthesis of the
proceed, albeit slowly. At IMP, the nucleotide in excess feedback
purine ring.
inhibits its own synthesis, thus allowing the partner purine
PRPP amidotransferase is regulated partly by GMP and partly by

AMP. The presence of either of these can reduce the enzymes
activity. Only when both are present is the enzyme fully
nucleotide to be made and balance to be achieved. When both

nucleotides are in abundance, then PRPP amidotransferase is
fully inhibited and the production of purines is stopped, thus
preventing them from over-accumulating.
inactivated. Subsequent reactions include adding glycine, adding

carbon (from N10-formyltetrahydrofolate), adding amine (from
glutamine), closing of the first ring, addition of carboxyl (from
CO2), addition of aspartate, loss of fumarate (a net gain of an
amine), addition of another carbon (from N10formyltetrahydrofolate), and closing of the second ring to form
Deoxyribonucleotide de novo Synthesis

Synthesis of deoxyribonucleotides de novo requires an interesting
enzyme called ribonucleotide reductase (RNR). RNR catalyzes
the formation of deoxyribonucleotides from ribonucleotides. The
inosine monophosphate (IMP).

IMP is a branch point for the synthesis of the adenine and
guanine nucleotides. The pathway leading from IMP to AMP
involves addition of amine from asparate and requires energy
from GTP. The pathway from IMP to GMP involves an oxidation
and addition of an amine from glutamine. It also requires energy
from ATP. The pathway leading to GMP is inhibited by its end
product and the pathway to AMP is inhibited by its end product.
Thus, balance of the purine nucleotides is achieved from the IMP
branch point forward. It is at this point that the significance of the
unusual regulation of PRPP amidotransferase becomes apparent.
If there is an imbalance of AMP or GMP, the enzyme is slowed,
dNTP de novo Synthesis

192
most common form of RNR is the Type I enzyme, whose
When a
substrates are ribonucleoside diphosphates (ADP, GDP, CDP, or
deoxypyrimidine
UDP) and the products are deoxyribonucleoside diphosphates
triphosphate, dTTP is
(dADP, dGDP, dCDP, or dUDP). Thymidine nucleotides are
abundant, it binds to
synthesized from dUDP. RNR has two pairs of two identical
the specificity site
subunits - R1 (large subunit) and R2 (small subunit). R1 has two
and inhibits binding
allosteric binding sites and an active site. R2 forms a tyrosine
and reduction of
radical necessary for the reaction mechanism of the enzyme.
pyrimidine
Because a single enzyme, RNR, is responsible for the synthesis of

all four deoxyribonucleotides, it is necessary to have mechanisms
to ensure that the enzyme produces the correct amounts of each
dNDP. This means that the enzyme must be responsive to the
levels of the each deoxynucleotide, selectively making more of
those that are in short supply, and preventing synthesis of those
that are abundant. These demands are met by having two
separate control mechanisms, one that determines which
substrate will be acted on, and another that controls the enzymes
catalytic activity.
Ribonucleotide reductase is allosterically regulated via two
binding sites - a specificity binding site (binds dNTPs and
controls which substrates the enzyme binds and thus, which
deoxyribonucleotides are made) and an activity binding site
(controls whether or not enzyme is active - ATP activates, dATP
inactivates).
diphosphates (CDP
and UDP) but
stimulates binding
and reduction of GDP
by the enzyme.
Conversely, binding
of the deoxypurine
triphosphate, ATP
stimulates reduction
of pyrimidine
diphosphates, CDP
and UDP.
Students sometimes
confuse the active
site of RNR with the
activity site. The
active site is where
the reaction is
Formation of Thymidine Nucleotides

193
catalyzed, and could also be called the catalytic site, whereas the
activity site is the allosteric binding site for ATP or dATP that
controls whether the enzyme is active.
Synthesis of dTTP by the de novo pathway takes a convoluted
pathway from dUDP to dUTP to dUMP to dTMP, then dTDP, and
finally dTTP. Conversion of dUMP to dTMP, requires a
tetrahydrofolate derivative and the enyzme thymidylate synthase.
In the process, dihydrofolate is produced and must be converted
back to tetrahyrdolate in order to keep nucleotide synthesis
occurring. The enzyme involved in the conversion of dihydrofolate
to tetrahydrofolate, dihydrofolate reductase (DHFR), is a target of
anticancer drugs like methotrexate or aminopterin, which inhibit
the enzyme,.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
194
Chapter 8
Signaling
Cells must receive and respond to signals from their

surroundings. Cellular signals and the pathways
through which they are passed on and amplified to
produce the desired eects on their targets are the
focus of this section.
Signaling
Cell Signaling
Cell Signaling
Ligand-gated Ion Channel Receptors
How do cells receive signals from their environment and how do they
Nuclear Hormone Receptors
communicate among themselves? It is intuitively obvious that even bacterial cells
G-protein Coupled Receptors (GPCRs)

Receptor Tyrosine Kinases (RTKs)
must be able to sense features of their environment, such as the presence of

nutrients or toxins, if they are to survive. In addition to being able to receive
information from the environment, multicellular organisms must find ways by which
their cells can communicate among themselves. Since dierent cells take on
specialized functions in a multicellular organism, they must be able to coordinate
activities perfectly like the musicians in an orchestra performing a complicated
piece of music. Cells grow, divide, or dierentiate in response to specific signals.
They may change shape or migrate to another location. At the physiological level,
cells in a multicellular organism, must respond to everything from a meal just eaten
to injury, threat or the availability of a mate. They must know when to repair
damage to DNA, when to undergo apoptosis (programmed cell death) and even
when to regenerate a lost limb. A variety of mechanisms have arisen to ensure
that cell-cell communication is not only possible, but astonishingly swift, accurate
and reliable.
How are signals sent between cells?
Like pretty much everything that happens in

on Signaling Mechanisms
HERE and HERE
cells, signaling is dependent on molecular

196
recognition. The basic principle of cell-cell signaling is simple. A
combinations of signals. The binding of a signal molecule to a
particular kind of molecule, sent by a signaling cell, is recognized
receptor sets o a chain of events in the target cell. These events
and bound by a receptor protein in (or on the surface of) the
could cause change in various ways, including, but not limited to,
target cell. The signal molecules are chemically varied- they may
alterations in metabolic pathways or gene expression in the target
be proteins, short peptides, lipids, nucleotides or catecholamines,
cell.
to name a few. The chemical properties of the signal determine

whether its receptors are on the cell surface or intracellular. If the
signal is small and hydrophobic it can cross the cell membrane
and bind a
How the binding of a signal to a receptor brings about change in

cells is the topic of this section. Although the specific molecular
components of the various signal transduction pathways dier,
they all have some
receptor inside the
features in common:
cell. If, on the

other hand, the
The binding of a signal
signal is charged,
to its receptor is usually,
or very large, it
though not always,
would not be able
followed by the
to diuse through
generation of a new
the plasma
signal(s) within the cell.
membrane. Such
signals need
Cellular Signaling
The process by which

the original signal is
receptors on the cell surface, typically transmembrane proteins
converted to a dierent form and passed on within the cell to
that have an extracellular portion that binds the signal and an
bring about change is called signal transduction.
intracellular part that passes on the message within the cell.

Most signaling pathways have multiple signal transduction steps
Receptors are specific for each type of signal, so each cell has
by which the signal is relayed through a series of molecular
many dierent kinds of receptors that can recognize and bind the
messengers that can amplify and distribute the message to
many signals it receives. Because dierent cells have dierent
various parts of the cell.
sets of receptors, they respond to dierent signals or

197
The last of these messengers usually interacts
Talking on a Cell Phone
with a target protein(s) and changes its activity,
A little cell was waiting for a message from its friend

It couldn't text or Facebook, no e-mails could it send.
What will it do, you wonder, how will it get a clue
About the world around it and what it needs to do?
What languages are spoken by cells that have no voice?
How do they know which way to go when they must make a choice?
Cells use many signals, molecules galore
Arriving at a target cell, some slip in through the door.
Hydrophobic signals through the plasma membrane slide
They're greeted by receptors on the cytoplasmic side.
But if they're hydrophilic on the membrane they must find
A cell-surface receptor to which they soon can bind.
often by phosphorylation.
When a signal sets a particular pathway in
motion, it is acting like an ON switch. This
means that once the desired result has been
obtained, the cell must have a mechanism that
acts as an OFF switch.
Understanding this underlying similarity is
helpful, because learning the details of the
dierent pathways becomes merely a matter of
identifying which molecular component
performs a particular function in each individual
Receptors binding signals will cause a change in cells

Sometimes a gene is turned on, sometimes the message tells
A kinase to phosphorylate and start a big cascade
To nudge awake some enzymes, whose actions can pervade
The metabolic pathways and change the cell's routine
To death or cell division and everything between.
case. We will consider several dierent signal

transduction pathways, each mediated by a
dierent kind of receptor. The first two
examples we will examine are those with the
fewest steps between the binding of the signal
by a receptor and a cellular response.
So cells have mechanisms to help communicate

To share a little gossip about their inner state
To tell a friend who's far away an enzyme to secrete
They do this all discreetly without the need to tweet
The chattiest of humans lie silent when in bed
But cells are always talking, unless, of course, they're dead.
198
Ligand-gated Ion Channel Receptors

The simplest and fastest of signal pathways is seen in the case of
signals whose receptors are gated ion channels. Gated ion
channels are made up of multiple transmembrane proteins that
create a pore, or channel, in the cell membrane. Depending upon
its type, each ion channel is specific to the passage of a particular
ionic species. The term "gated" refers to the fact that the ion
channel is controlled by a "gate" which must be opened to allow
the ions through. The gates are opened by the binding of an
respond to a message from the

neighboring nerve cell. The nerve
cell releases a neurotransmitter
signal into the synaptic cleft, which is
the space between the nerve cell
and the muscle cell it is "talking to".
Examples of neurotransmitter signal
molecules are acetylcholine and
serotonin, shown above.
Neurotransmitters
incoming signal (ligand) to the receptor, allowing the almost
When acetylcholine molecules are released into the synaptic cleft
instantaneous passage of millions of ions from one side of the
(the space between the pre- and post-synaptic cells) they diuse
membrane to the
rapidly till they reach their receptors on the membrane of the
other. Changes in
muscle cell. The
the interior
binding of the
environment of the
acetylcholine to its
cell are thus
receptor, an ion
brought about in
channel on the
microseconds and
membrane of the
in a single step.
muscle cell,
This type of swift

response is seen,
for example, in
neuromuscular
junctions, where
Signaling Through Gated Ion Channels
muscle cells
causes the gate in

the ion channel to
open. The
resulting ion flow
through the
channel can
immediately
Signaling Across Nerve Cells

199
change the membrane potential. This, in turn, can trigger other
incapable of crossing the plasma membrane, and thus, must have
changes in the cell. The speed with which changes are brought
cell surface receptors.
about in neurotransmitter signaling is evident when you think

about how quickly you remove your hand from a hot surface.
Sensory neurons carry information to the brain from your hand on
the hot surface and motor neurons signal to your muscles to
move the hand, in less time than it took you to read this sentence!
Nuclear Hormone Receptors
By contrast, steroid hormones have receptors inside the cell

(intracellular receptors). Steroid hormone receptors are proteins
that belong in a family known as the nuclear receptors.
Nuclear hormone receptors are proteins with a double life: they
are actually dormant transcription regulators. In the absence of
signal, these receptors are in the cytoplasm, complexed with
Another type of relatively simple, though much slower, signaling is
other proteins (HSP in the figure on the following page) and
seen in pathways in which the signals are steroid hormones, like
inactive. When a steroid hormone enters the cell, the nuclear
estrogen or testosterone, pictured below. Steroid hormones, as
hormone receptor binds the hormone and dissociates from the
you are aware, are related to cholesterol, and as hydrophobic
HSP. The receptors, then, with the hormone bound, translocate
molecules, they are able to cross the cell membrane by
into the nucleus.
themselves. This is unusual, as most signals coming to cells are
In the nucleus, they regulate the transcription of target genes by

binding to their regulatory sequences (labeled HRE for hormoneresponse elements). The binding of the hormone-receptor
complex to the regulatory elements of hormone-responsive genes
modulates their expression. Because these responses involve
gene expression, they are
relatively slow.
Most other signaling pathways,
besides the two we have just

HERE and HERE
discussed, involve multiple
steps in which the original signal is passed on and amplified

200
through a number of intermediate steps, before the cell responds
While the specific details of the signaling pathways that follow the
to the signal.
binding of signals to each of these receptor types are dierent, it
We will now consider two signaling pathways, each mediated by

a major class of cell surface receptor- the G-protein coupled
receptors (GPCRs) and the receptor tyrosine kinases (RTKs).
is easier to learn them when you can see what the pathways have
in common, namely, interaction of the signal with a receptor,
followed by relaying the signal through a variable number of
intermediate molecules, with the last of these molecules
interacting with target protein(s) to modify their activity in the cell.
G-protein
Coupled
Receptors
(GPCRs)
G-protein coupled
receptors are involved in
responses of cells to many
dierent kinds of signals,
from epinephrine, to
odors, to light. In fact, a
variety of physiological
phenomena including
vision, taste, smell and the
fight-or-flight response are
mediated by GPCRs.
What are G-protein
coupled receptors?
Steroid Hormones Act by Modulating Expression of Hormone-responsive Genes
G-protein coupled
201
receptors are cell surface receptors that pass on the signals that
extracellular domain of a GPCR, the receptor undergoes a
they receive with the help of guanine nucleotide binding proteins
conformational change that allows it to interact with a G-protein
(a.k.a. G-proteins). Before thinking any further about the signaling
that will then pass the signal on to other intermediates in the
pathways downstream of GPCRs, it is necessary to know a few
signaling pathway.
important facts about these receptors and the G-proteins that

assist them.
What is a G-protein?
As noted
Though there are hundreds of dierent G-protein coupled
above, a G-
receptors, they all have the same basic structure:
protein is a
they all consist of a single polypeptide chain that threads back
guanine
and forth seven times through the lipid bilayer of the plasma
nucleotide-
membrane. For this reason, they are sometimes called seven-
binding
pass transmembrane (7TM) receptors.
protein that
can interact
One end of the
with a G-
polypeptide
protein linked
forms the
receptor. G-
extracellular
proteins are
domain that
associated
binds the signal
with the
while the other
cytosolic side
end is in the
of the plasma
cytosol of the
membrane,
cell.
where they
When a ligand
(signal) binds the
G-protein Coupled Receptor
are ideally
G-protein Coupled Receptor Signaling
202
situated to interact with the cytosolic tail of the

GPCR, when a signal binds to the GPCR.
There are many dierent G-proteins, all of which
happens when a signal arrives at the cell surface and

See Kevins YouTube lectures binds to a GPCR.
The binding of a signal molecule by the extracellular
HERE and HERE
share a characteristic structure- they are
part of the G-protein linked receptor causes the
composed of three subunits called alpha, beta

and gamma (). Because of this, they are sometimes called
heterotrimeric G proteins (hetero=dierent, trimeric= having three
parts). The
the conformation of, a G-protein. This has two consequences:

First, the alpha
subunit of the G-
subunit of such
protein loses its GDP
proteins can bind
and binds a GTP
GDP or GTP and
instead.
is capable of
Second, the G-
hydrolyzing a
protein breaks up
GTP molecule
into the GTP-bound
bound to it into
part and the
GDP. In the
part.
unstimulated
state of the cell,
cytosolic tail of the receptor to interact with, and alter
G-protein with GDP Bound
that is, in the

absence of a signal bound to the GPCR, the G-proteins are found
in the trimeric form ( bound together) and the subunit has a
GDP molecule bound to it.
With this background on the structure and general properties of
the GPCRs and the G-proteins, we can now look at what
These two parts can

diuse freely along
the cytosolic face of
the plasma
membrane and act
upon their targets.
What happens
when G-proteins
interact with their
G-protein Activation
203
target proteins?
That depends on what the target is. G-proteins interact with
dierent kinds of target proteins, of which we will examine two
major categories:
Two well-studied examples of enzymes whose activity is

regulated by a G-protein are adenylate cyclase and
phospholipase C. When adenylate cyclase is activated, the
molecule cAMP is produced in large amounts.
Ion Channels
We have earlier seen that some gated ion channels can be
opened or closed by the direct binding of neurotransmitters to a
receptor that is an ion-channel protein. In other cases, ion
channels are regulated by the binding of G-proteins. That is,
instead of the signal directly binding to the ion channel, it binds to
a GPCR, which activates a G-protein that then binds and opens
Synthesis of cAMP
the ion channel. The change in the distribution of ions across the
plasma membrane causes a change in the membrane potential.
When phospholipase C is activated, the molecules inositol
Specific Enzymes
trisphosphate (IP3) and diacylglycerol
The interaction of G-proteins with
(DAG) are made. cAMP, IP3 and
their target enzymes can regulate the
DAG are second messengers, small,
activity of the enzyme, either
diusible molecules that can "spread
increasing or decreasing its activity.
the message" brought by the original
Often the target enzyme will pass
signal, to other parts of the cell.
the signal on in another form to
In these cases, the binding of a
another part of the cell. As you might
signal to the GPCR activated a G-
imagine, this kind of response takes
protein, which in turn, activated an
a little longer than the kind where an

ion channel is opened
instantaneously.
Second Messengers
enzyme that makes a second

messenger that can amplify the
message in the cell.
204
Protein Kinase A Activation
G-protein Signaling Cycle

We will first trace the eects of activating adenylate cyclase and
subunits that are bound tightly together. Upon binding of

cAMP the catalytic subunits are released from the
the resulting increase in cAMP.
regulatory subunits,
What is the eect of elevated cAMP levels?
allowing the enzyme to carry out its function, namely
cAMP molecules bind to, and activate an enzyme, protein kinase
phosphorylating other proteins.
A (PKA). PKA is composed of two catalytic and two regulatory

205
Thus, cAMP can
breakdown of glycogen, releasing glucose (in the form of
regulate the activity of
glucose-1-phosphate) for use by the cell. Changes in gene
PKA, which in turn, by
expression, likewise, lead to changes in the cell by altering the
phosphorylating other
production of particular proteins in response to the signal.
proteins can change
Although the steps described above seem complicated, they
their activity. The
follow the simple pattern outlined at the beginning of this section:
targets of PKA may be

enzymes that are
Binding of signal to receptor
activated by
Several steps where the signal is passed on through
phosphorylation, or
intermediate molecules (G-proteins, adenylate cyclase, cAMP,
they may be proteins
G-protein Nucleotide Swapping
and finally, PKA)
that regulate
Phosphorylation of target proteins by the kinase, leading to
transcription. The phosphorylation of a transcriptional activator,
changes in the cell.
for example, may cause the activator to bind to a regulatory

sequence on DNA and to increase the transcription of the gene it
Finally, if the signal binding to the receptor serves as a switch that
controls. The activation of previously inactive enzymes alters the
sets these events in motion, there must be mechanisms to turn
state of the cell by changing the reactions that are occurring
the pathway o. The first is at the level of the G-protein. Recall
within the cell.
that the alpha subunit of the G-protein is in its free and activated
state when it has GTP bound and that it associates with the beta-
For example, the binding of epinephrine to its receptor on the cell
gamma subunits and has a GDP bound when it is inactive. We
surface, activates, through the
also know that the alpha subunit
action of G-proteins, and
has an activity that enables it to
subsequent activation of PKA, the
hydrolyze GTP to GDP, as
phosphorylation of glycogen
shown in the figure above left.
phosphorylase. The resulting
This GTP-hydrolyzing activity
activation of glycogen
makes it possible for the alpha
phosphorylase leads to the
cAMP Breakdown
subunit, once it has completed

206
its task, to return to its GDP bound state, re-associate with

the beta-gamma part and become inactive again.
The second "o switch" is further down the signaling
pathway, and controls the level of cAMP. We just noted that
cAMP levels increase when adenylate cyclase is activated.
When its job is done, cAMP is broken down by an enzyme
called phosphodiesterase. When cAMP levels drop, PKA
returns to its inactive state, putting a halt to the changes
brought about by the activation of adenylate cyclase by an
activated G-protein.
Let us now examine the events that follow the activation of
Phospholipase C (PLC) by a G-protein. As we noted earlier,
the activation of PLC results in the production of the second
messengers IP3 and DAG. What do these molecules do?
Signaling Outcomes
The IP3 and DAG produced by activated phospholipase C work

together to activate a protein kinase. First, IP3 diuses to the
endoplasmic reticulum membrane where it binds to gated
calcium ion channels. This causes calcium channels in the ER
membrane to open and release large amounts of calcium into the
cytoplasm from the ER lumen, as
shown in the figure below.
The increase in cytosolic calcium
Phospholipase C Signaling
ion concentration has various

HERE and HERE
eects, one of which is to

207
activate a protein kinase

called protein kinase C (C
Gee, I Wish I Could Do That
for calcium), together with

the DAG made in the
earlier step. Like PKA,
Protein kinase C
phosphorylates a variety
of proteins in the cell,
altering their activity and
thus changing the state of
the cell.
The pathways leading to
PKC and PKA activation
following the binding of a
signal to a GPCR are
summarized above.
Receptor
Tyrosine Kinases
Receptor tyrosine kinases
mediate responses to a
large number of signals,
including peptide
GPCRs, as you know,

Have a G-protein in tow.
Alpha, beta, gamma, sit
Waiting till the signals hit.
Another enzyme, PLC,

Awaits an alpha-GTP
To activate it, so it splits
PIP2, and makes two bits.
When receptors signals bind,

G-proteins respond in kind,
Swapping out their GDP
For triphosphate gleefully.
IP3 and DAG

Are second messengers, you see.
They work together as a team,
They've got a cunning little scheme.
Alpha, with its GTP,

Leaves all full of energy.
A cyclase it will activate
That acts upon adenylate.
IP3 at ion gates

On ER membranes lets a spate
Of calcium into cytosol
That's bad enough, but that's not all.
Cyclic AMPs then find

A PKA that they can bind.
With binding of cAMPs
R subunits part from Cs.
The calcium binds a PKC,

And with the help of DAG,
The kinase it can activate
Proteins to phosphorylate.
C subunits, floating free,

Are on a phosphate adding spree.
PKA phosphorylates
And target proteins activates
The binding of a signal small

To GPCRs, caused this all.
Transducing pathways work a spell
And change the actions of a cell.
hormones like insulin and

growth factors like
epidermal growth factor.

208
Like the GPCRs, receptor tyrosine kinases bind a signal, then
tyrosine kinase activity of these tails
pass the message on through a series of intracellular molecules,
to be turned on. The activated tails
the last of which acts on target proteins to change the state of the
then phosphorylate each other on
cell.
several tyrosine residues. This is

As the name suggests, a receptor
Receptor Tyrosine Kinase

before signal binding
called autophosphorylation.
tyrosine kinase is a cell surface
The phosphorylation of tyrosines on
receptor that also has a tyrosine
the receptor tails triggers the
kinase activity. The signal binding
assembly of an intracellular signaling
domain of the receptor tyrosine
complex on the tails. The newly
kinase is on the cell surface, while the
phosphorylated tyrosines serve as
tyrosine kinase enzymatic activity
binding sites for signaling proteins
resides in the cytoplasmic part of the
that then pass the message on to yet
protein (see figure above). A
other proteins. An important protein that is subsequently
transmembrane alpha helix connects
activated by the signaling complexes on the receptor tyrosine
these two regions of the receptor.
kinases is called Ras.
Activated tyrosine
kinase domains add
phosphate onto each
other
What happens when signal

molecules bind to receptor
tyrosine kinases?
The Ras protein is a monomeric guanine nucleotide binding
Binding of signal molecules to the
cytosolic face of the
protein that is
associated with the
plasma membrane (in
extracellular domains of receptor
fact, it is a lot like the
tyrosine kinase molecules causes two

receptor molecules to dimerize (come
together and associate). This brings
the cytoplasmic tails of the receptors
close to each other and causes the
Signal binding causes

dimerization of receptor and
activation of tyrosine kinase
domains
alpha subunit of trimeric

Complex of signaling proteins
assembles on phosphorylated
RTK tails. This complex can
activate Ras.
G-proteins). Just like the

alpha subunit of a Gprotein, Ras is active
209
genes are expressed. The combined eect of changes in gene

expression and protein activity alter the cell's physiological state.
Once again, in following the path of signal transduction mediated
by RTKs, it is possible to discern the same basic pattern of
events: a signal is bound by the extracellular domains of receptor
tyrosine kinases, resulting in receptor dimerization and
Ras Activation
when GTP is bound to it and inactive when GDP is bound to it.

Also, like the alpha subunit, Ras can hydrolyze the GTP to GDP.
When a signal arrives at the receptor tyrosine kinase, the receptor
monomers come together and phosphorylate each others'
tyrosines, triggering the assembly of a complex of proteins on the
autophosphorylation of the cytosolic tails, thus conveying the

message to the interior of the cell.
The message is passed on via a signalling complex to Ras
which then stimulates a series of kinases. The terminal kinase in
the cascade acts on target proteins and brings about in changes
in protein activities and gene expression.
cytoplasmic tail of the receptor. One of the proteins in this

complex interacts with Ras and stimulates the exchange of the
GDP bound to the inactive Ras for a GTP. This activates the Ras.
Activated Ras triggers a phosphorylation cascade of three protein
kinases, which relay and distribute the signal. These protein
kinases are members of a group called the MAP kinases (Mitogen
Activated Protein Kinases). The final kinase in this cascade
phosphorylates various target proteins, including enzymes and
transcriptional activators that regulate gene expression.
The phosphorylation of various enzymes can alter their activities,
and set o new chemical reactions in the cell, while the
phosphorylation of transcriptional activators can change which
Activated Ras Cascade

210
The descriptions above provide a very simple sketch of some of
The Tao of Hormones
the major classes of receptors and deal primarily with the
To the tune of "The Sound of Silence"
mechanistic details of the steps by which signals received by

various types of receptors bring about changes in cells. A major
Biochemistry my friend
It's time to study you again
Mechanisms that I need to know
Are the things that really stress me so
"Get these pathways planted firmly in your head,"
Ahern said
Let's start with ep-inephrine

Membrane proteins are well known
Changed on binding this hormone
Rearranging selves without protest
Stimulating a G alpha S
To go open up and displace its GDP
With GTP
Because of ep-inephrine
Active G then moves a ways
Stimulating ad cyclase
So a bunch of cyclic AMP
Binds to kinase and then sets it free
All the active sites of the kinases await
Triphosphate
Muscles are affected then
Breaking down their glycogen
So they get a wad of energy
In the form of lots of G-1-P
And the synthases that could make a glucose chain
All refrain
Now I've reached the pathway end
Going from adrenalin
Here's a trick I learned to get it right
Linking memory to flight or fright
So the mechanism that's the source of anxious fears
Reappears
When I make ep-inephrine
take-home lesson is the essential similarity of the dierent

pathways.
Another point to keep in mind is that while we have looked at
each individual pathway in isolation, a cell, at any given time
receives multiple signals that set o a variety of dierent
responses at once. The pathways described above show a
considerable degree of "cross-talk" and the response to any
given signal is aected by the other signals that the cell receives
simultaneously. The multitude of dierent receptors, signals and
the combinations thereof are the means by which cells are able to
respond to an enormous variety of dierent circumstances.

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211
Chapter 9
Techniques
In this section, we describe some of the methods

biochemists use to do their work.
Techniques
Introduction
Introduction
Cell Disruption
The environment of a cell is very complex, making it very dicult, if not impossible,
Fractionation
Ion Exchange Chromatography
Gel Exclusion Chromatography
Anity Chromatography
HPLC
Histidiine Tagging
to study individual reactions, enzymes, or pathways within it. For this reason,
biochemists prefer to isolate molecules (enzymes, DNAs, RNAs, and other
molecules of interest) so they can be analyzed without interference from the
millions of other processes occurring simultaneously in the cell. Many of the
methods used in isolating molecules from cells involve some form of
Electrophoresis
chromatography. To separate compounds from their cellular environments, one
Agarose
must first break open (lyse) the cells.
SDS-PAGE
Isoelectric Focusing
Cell Disruption
2-D Gel Electrophoresis
There are several ways to break open cells. Lysis methods include lowering the
Protein Cleavage
Microarrays
Blotting
Making Recombinant DNAs
PCR
Lac Z Blue-White Screening
Reverse Transcription
ionic strength of the medium cells are kept in. This can cause cells to swell and
burst. Mild surfactants may be used to enhance the eciency of lysis. Most
bacteria, yeast, and plant tissues, which have cell
walls, are resistant to such osmotic shocks,
however, and stronger disruption techniques are
often required.

Kevins Techniques lectures
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213
Enzymes may be useful in helping to degrade the cell walls.
processed to separate the molecules into smaller subsets, or
Lysozyme, for example, is very useful for breaking down bacterial
fractions.
walls. Other enzymes commonly employed include cellulase

(plants), glycanases, proteases, mannases, and others.
Mechanical agitation may be employed in the form of tiny beads
that are shaken with a suspension of cells. As the beads
bombard the cells at high speed, they break them open.
Sonication (20-50 kHz sound waves) provides an alternative
method for lysing cells. The method is noisy, however, and
generates heat that can be problematic for heat-sensitive
compounds.
Fractionation
Fractionation of samples typically starts with centrifugation.
Using a centrifuge, one can remove cell debris, and fractionate
organelles, and cytoplasm. For example, nuclei, being relatively
large, can be spun down at fairly low speeds. Once nuclei have
been sedimented, the remaining solution, or supernatant, can be
centrifuged at higher speeds to obtain the smaller organelles, like
mitochondria. Each of these fractions will contain a subset of the
Another means of disrupting cells involves using a cell bomb.

In this method, cells are placed under very high pressure (up to
25,000 psi). When the pressure is released, the rapid pressure
change causes dissolved gases in cells to be released as bubbles
which, in turn, break open the cells.
Cryopulverization is often employed for samples having a tough
extracellular matrix, such as connective tissue or seeds. In this
technique, tissues are flash-frozen using liquid nitrogen and then
ground to a fine powder before extraction of cell contents with a
buer.
Whatever method is employed, the crude lysates obtained
contain all of the molecules in the cell, and thus, must be further
Fractionation by Centrifugation
214
molecules in the cell. Although every subset contains fewer
the support consists of tiny beads to which are attached
molecules than does the crude lysate, there are still many
chemicals possessing a charge. Each charged molecule has a
hundreds of molecules in each. Separating the molecule of
counter-ion. The figure shows the beads (blue) with negatively
interest from the others is where chromatography comes into
charged groups (red) attached. In this example, the counter-ion is
play. We will consider several separation techniques.
sodium, which is positively charged. The negatively charged
Many chromatographic techniques

are performed in columns.
These are tubes containing the
material (called the support) used
to perform the separation .
Supports are designed to exploit
the chemical, or size, dierences of
the many molecules in a mixture.
Columns are packed (filled) with
the support and a buer or solvent
carries the mixture of compounds
to be separated through the
support. Molecules in the sample
interact dierentially with the
support and consequently, will
travel through it with dierent
speeds.
Ion Exchange
Chromatography
In ion exchange chromatography,
Cation Exchange Chromatography

215
groups are unable to leave the
Gel Exclusion
Chromatography
beads, due to their covalent

attachment, but the counter-
Gel exclusion
ions can be exchanged for
chromatography (also
molecules of the same charge.
called molecular exclusion
Thus, a cation exchange
chromatography, size
column will have positively
exclusion chromatography,
charged counter-ions and
or gel filtration
positively charged compounds
chromatography) is a low
present in a mixture passed
resolution isolation
through the column will
method that employs a
exchange with the counter-ions
cool trick. This involves
and stick to the negatively
the use of beads that have
charged groups on the beads.
tiny tunnels in them that
Molecules in the sample that
each have a precise size.
are neutral or negatively
The size is referred to as
charged will pass quickly

through the column. On the
other hand, in anion exchange
an exclusion limit, which

Gel Exclusion Chromatography
chromatography, the chemical groups attached to the beads are

positively charged and the counter-ions are negatively charged.
Molecules in the sample that are negatively charged will stick
and other molecules will pass through quickly. To remove the
molecules stuck to a column, one simply needs to add a high
concentration of the appropriate counter-ions to displace and
release them. This method allows the recovery of all components
of the mixture that share the same charge.
means that molecules

above a certain molecular
weight will not fit into the tunnels. Molecules with sizes larger
than the exclusion limit do not enter the tunnels and pass through
the column relatively quickly by
making their way between the
beads. Smaller molecules,
which can enter the tunnels, do
so, and thus, have a longer path

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216
Anity Chromatography
Anity chromatography is a very powerful technique
that exploits the binding anities of target molecules
(typically proteins) for substances covalently linked
to beads. For example, if one wanted to separate all
of the proteins in a sample that bound to ATP from
proteins that do not bind ATP, one could covalently
link ATP to support beads and then pass the sample
through column. All proteins that bind ATP will
stick to the column, whereas those that do not
bind ATP will pass quickly through it. The proteins
adhering to the column may then released from the
column by adding ATP.
High Performance Liquid

Chromatography (HPLC)
HPLC (also sometimes called High Pressure Liquid
Chromatography) is a powerful tool for separating
Affinity Chromatography
that they take in passing through the column. Because of this,
smaller molecules based on their dierential

polarities. It employs columns with supports made
of very tiny beads that are so tightly packed that flow
molecules larger than the exclusion limit will leave the column
of solvents/buers through the columns requires the application
earlier, while those that pass through the beads will elute from the
of high pressures (hence the name). The supports used can be
column later. This method allows separation of molecules by
polar (normal phase separation) or non-polar (reverse phase
their size.
separation). In normal phase separations, non-polar molecules

elute first followed by the more polar compounds. This order is
217
(usually six) to the coding sequence for a protein. The protein

produced when this gene is expressed has a run of histidine
residues fused at either the carboxyl or amino terminus to the
amino acids in the remainder of the protein. The histidine side
chains of this tag have an anity for nickel or cobalt ions,
making separation of histidine-tagged proteins from a cell lysate
is relatively easy. Simply passing the sample through a column
that has immobilized nickel or cobalt ions allows the histidinetagged proteins to stick, while the remaining cell proteins all
pass quickly through. The histidine-tagged proteins are then
eluted by addition of imidazole (which is chemically identical to
the histidine side chain) to the column. Histidine tags can be
cleaved o using endopeptidases.
Electrophoresis
Histidine Tagging
switched in reverse phase chromatography. Of the two, reverse
DNA molecules are long and loaded with negative charges,

thanks to their phosphate backbones. Electrophoretic methods
separate large
phase is much more commonly employed to due more
molecules, such as DNA,
reproducible chromatographic profiles (separations) that it
RNA, and proteins based
typically produces.
on their charge and size.

For DNA and RNA, the
Histidine Tagging
Histidine tagging is a powerful tool for isolating a recombinant
protein from a cell lysate. It relies on using recombinant DNA
techniques to add codons specifying a series of histidines
charge of the nucleic acid

is proportional to its size
(length). For proteins,
which do not have a
DNA Fragments Separated by

Agarose Gel Electrophoresis
218

Agarose gel electrophoresis is a method for separating nucleic
acids. It is worth noting
that nucleic acids are
the largest molecules
found in cells, in some
cases by orders of
magnitude. Agarose
provides a matrix which
encases a buer. The
matrix provides
openings for
macromolecules to
move through and the
largest macromolecules
have the most dicult
time navigating, whereas
SDS-PAGE Separation of Proteins
the smallest
macromolecules slip through it the easiest. Unlike column
chromatography, electrophoresis uses an electric current as a
force to drive the molecules through the matrix. Since the size to
uniform charge, a clever trick is employed to make them mimic

nucleic acids.
charge ratios for DNA and RNA are constant for all sizes of these
nucleic acids, the size per force is also constant (since force is
directly proportional to charge), so the molecules simply sort on
the basis of their size - the smallest move fastest and the largest
move slowest. Visualization of the DNA fragments in the gel is
219
made possible by addition of a dye, such
eective in separating smaller molecules,
as ethidium bromide that fluoresces
whereas lower percentages of acrylamide
under ultraviolet light.
reverse that. Second, proteins must be

physically altered to present themselves to
the matrix like the negatively charged rods of
SDS-Polyacrylamide Gel
Electrophoresis (SDSPAGE)
DNA. This is accomplished by treating the
Like DNA and RNA, proteins are large
molecules coat the proteins such that the
macromolecules. Proteins, however, vary
exterior surface is loaded with negative
tremendously in their charge. Whereas
charges proportional to the mass, just like the
double-stranded DNA is rod-shaped,
backbone of DNA. Third, a stacking gel may
most proteins are globular (folded up).
be employed at the top of the gel to provide a
Further, proteins are considerably smaller
way of compressing the samples into a tight
than nucleic acids, so the openings of
band before they enter the main
the matrix of the agarose gel are simply
polyacrylamide gel (called the resolving gel).
too large to eectively provide
proteins with the detergent called SDS (sodium

dodecyl sulfate). SDS denatures the proteins
so they assume a rod-like shape and the SDS
2D Gel Electrophoresis
separation. Consequently, unlike nucleic
Just as DNA fragments get sorted on the basis

of size (largest move slowest and smallest
acids, proteins cannot be eectively separated by electrophoresis
move fastest), the proteins migrate through the gel matrix at rates
on agarose gels. To separate proteins by electrophoresis, one
inversely related to their size. Upon completion of the
must make several modifications. First, a matrix made by
electrophoresis, there are several means of staining to visualize
polymerizing and crosslinking acrylamide units is
the proteins on the gel. They include reagents, such
employed. One can adjust the pore size of the

matrix readily by changing the percentage of
acrylamide in the gel. Higher percentages of
acrylamide create smaller pores and are more
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as Coomassie Brilliant Blue or silver nitrate (the

latter is much more sensitive than Coomassie Blue
staining and can be used when there are very small
quantities of protein).
220
Isoelectric Focusing
separate the proteins by their pI values. Next, as shown on the
Proteins vary considerably in their charges and, consequently, in
proteins is rotated through 90 and placed on top of a regular
their pI values (pH at which their charge is zero). Separating

proteins by isoelectric focusing requires establishment of a pH
gradient in an acrylamide gel matrix. The matrixs pores are
adjusted to be large to reduce the eect of sieving based on size.
Molecules to be focused are applied to the gel with the pH
gradient and an electric current is passed through it. Positively
previous page, the isoelectric gel containing the separated

polyacrylamide gel for SDS-PAGE analysis (to separate them
based on size). The proteins in the isoelectric gel matrix are
electrophoresed into the polyacrylamide gel and separation on
the basis of size is performed. The product of this analysis is a
2D gel, in which proteins are sorted by both mass and charge.
charged molecules, for example, move towards the negative
The power of 2D gel electrophoresis is that virtually every protein
electrode, but since they are traveling through a pH gradient, as
in a cell can be separated and appear on the gel as a distinct
they pass through it, they reach a region where their charge is
spot. In the figure, spots in the upper left correspond to large
zero and, at that point, they stop moving. They are at that point
positively charged proteins, whereas those in the lower right are
attracted to neither the positive nor the negative electrode and
small negatively charged ones. It is possible using high-
are thus focused at their pI. By using isoelectric focusing, it is
throughput mass spectrometry analysis to identify every spot on
possible to separate proteins whose pI values dier by as little as
a 2D gel. This is particularly powerful when one compares protein
0.01 units.
profiles between dierent tissues or between the samples of the
2D Gel Electrophoresis
Both SDS-PAGE and isoelectric focusing are powerful
techniques, but a clever combination of the two is a powerful tool
of proteomics - the science of studying all of the proteins of a
cell/tissue simultaneously. In 2D gel electrophoresis, an extract
containing the proteins is first prepared. One might, for example,
be studying the proteins of liver tissue. The liver cells are lysed
and all of the proteins are collected into a sample. Next, the
Protein Cleaving Agents
sample is subjected to isoelectric focusing as described earlier, to

221
same tissue treated or
Microarrays
untreated with a
2D gels are one way of surveying a broad spectrum of molecules
particular drug.
simultaneously. Other approaches to doing the same thing
Comparison of a 2D
involve what are called microarrays. DNA microarrays, for
separation of a non-
example, can be used to determine all of the genes that are being
cancerous tissue with a
expressed in a given tissue, simultaneously. Microarrays employ
cancerous tissue of the
a grid (or array) made of rows and columns on a glass slide, with
same type provides a
each box of the grid containing many copies of a specific
quick identification of
molecule, say a single-stranded DNA molecule corresponding to
proteins whose level of
the sequence of a single unique gene. As an example, consider
expression diers
scanning the human genome for all of the known mRNA
between them.
sequences and then synthesizing single stranded DNAs
Information such as this

might be useful in
Microarray
designing treatments or in
determining the mechanisms by which the cancer arose.
Protein Cleavage
Working with intact proteins in analytical techniques, such as
mass spectrometry, can be problematic. Consequently, it is often
desirable to break a large polypeptide down into smaller, more
manageable pieces. There are two primary approaches to
accomplishing this - use of chemical reagents or use of
proteolytic enzymes. The table on the previous page shows the
cutting specificities of various cleavage agents.
complementary to each mRNA. Each complementary DNA

sequence would have its own spot on the matrix. The position of
each unique gene sequence on the grid is known and the entire
grid would represent all possible genes that are expressed. Then
for a simple gene expression analysis, one could take a tissue
(say liver) and extract the mRNAs from it. These mRNAs
represent all the genes that are being expressed in the liver at the
time the extract was made. The mRNAs can easily be tagged
with a colored dye (say blue). The mixture of tagged mRNAs is
then added to the array and base-pairing conditions are created
to allow complementary sequences to find each other. When the
process is complete, each liver mRNA should have bound to its
corresponding gene on the array, creating a blue spot in that box
on the grid. Since it is known which genes are in which box, a
222
blue spot in a box indicates that the gene in that box was
used to study the binding of proteins or other molecules to the
expressed in the liver. The presence and abundance of each
peptides.
mRNA may then readily determined by measuring the amount of

blue dye at each box of the grid. A more powerful analysis could
Blotting
be performed with two sets of mRNAs, each with a dierent
Blotting provides a means of identifying specific molecules out of
colored tag (say blue and yellow). One set of mRNAs could come
a mixture. It employs three main steps. First, the mixture of
from the liver of a vegetarian (tagged blue) and the other from a
molecules is separated by gel electrophoresis. The mixture could
meat eater (tagged yellow),
be DNA (Southern Blot), RNA
for example. The mRNAs are
(Nothern Blot), or protein (Western
mixed and then added to the
Blot) and the gel could be agarose
array and complementary
(for DNA/RNA) or polyacrylamide
sequences are once again
(for protein). Second, after the gel
allowed to form duplexes.
run is complete, the proteins or
After unhybridized mRNAs
nucleic acids in the gel are
are washed away, the plate is
transferred out of the gel onto a
analyzed. Blue spots in grid
membrane/paper that physically
boxes correspond to mRNAs
binds to the molecules. This blot,
present in the vegetarian
as it is called, has an imprint of the
liver, but not in that of the
bands of nucleic acid or protein that
meat eater. Green spots

(blue plus yellow) would
Northern Blotting Procedure
were in the gel (see figure at left).

The transfer can be accomplished
correspond to mRNAs
by diusion or by using an electrical current to move the
present in equal abundance in the two livers. The intensity of
molecules from the gel onto the membrane. The membrane may
each spot would also give information about the relative amounts
be treated to covalently link the bands to the surface of the blot.
of each mRNA in the tissues. Similar analyses could be done,
Last, a visualizing agent specific for the molecule of interest in the
using cDNAs instead of mRNA. Peptide microarrays have
mixture is added to the membrane. For DNA/RNA, that might be
peptides bonded to the glass slide instead of DNA and can be

223
a complementary nucleic acid sequence that is

labeled in some fashion (radioactivity or dye). For a
protein, it would typically involve an antibody that
specifically binds to the protein of interest. The
bound antibody can then be targeted by another
antibody specific for the first antibody. The secondary
antibody is usually linked to an enzyme which, in the
presence of the right reagent, catalyzes a reaction
that produces a signal (color or light) indicating where
the antibody is bound. If the molecule of interest is in the original
mixture, it will light up and reveal itself.
Making a Recombinant
up the proper conditions for the protein to be made in the target

cells. For bacteria, this typically involves the use of plasmids.
Plasmids are circular, autonomously replicating DNAs found
Making Recombinant DNAs
commonly in bacterial cells. Plasmids used in recombinant DNA
Molecular biologists often create recombinant DNAs by joining
markers that allow researchers to identify cells carrying them
together DNA fragments from dierent sources. One reason for
(antibiotic resistance, for example) and 3) contain sequences
making recombinant DNA molecules is to enable the production
(such as a promoter and Shine Dalgarno sequence) necessary for
of a specific protein that is of
expression of the desired protein in the target cell. A plasmid that
interest. For example, it is possible
has all of these features is referred to as an expression vector
to engineer a recombinant DNA
(see an example in the figure at left). Plasmids may be extracted
molecule containing the gene for
from the host, and any gene of interest may be inserted into
human growth hormone and
them, before returning them to the host cell. Making such
introduce it into an organism like a
recombinant plasmids is a relatively simple process. It involves 1)
bacterium or yeast, which could
cutting the gene of interest with a restriction enzyme
make massive quantities of the
(endonucleases which cut at specific DNA sequences); 2) cutting
human growth hormone protein very
the expression plasmid DNA with restriction enzyme, to generate
cheaply. To do this, one needs to set
An Expression Vector
methods 1) replicate in high numbers in the host cell; 2) carry
ends that are compatible with the ends of the gene of interest;
224
3) joining the gene of interest to the plasmid DNA using DNA

ligase; 4) introducing the recombinant plasmit into a bacterial cell;
and 5) growing cells that contain the plasmid. The bacterial cells
bearing the recombinant plasmid may then be induced to express
the inserted gene and produce large quantities of the protein
encoded by it.
Polymerase Chain
Reaction (PCR)
PCR allows one to use the

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power of DNA replication to

obtain large amounts of a specific DNA in a short time. As
everyone knows, cell division results in doubling the number of
cells with each round of division. Each time cells divide, DNA
must be replicated, as well, so the amount of DNA is doubling as
the cells are doubling. Kary Mullis recognized this fact and came
up with the technique of PCR, which mimics DNA replication. In
contrast to cellular DNA replication, which amplifies all of a cells
DNA during a replication cycle, PCR is used to replicate only a
specific segment of DNA. This segment of DNA, known as the
target sequence, is replicated repeatedly, to obtain millions of
copies of the target. Just as in DNA replication, PCR requires a
template DNA, 4 dNTPs, primers to initiate DNA synthesis on
each strand, and a DNA polymerase to synthesize the new DNA
copies. In PCR, the primers bind to sequences flanking the target
region that is to be amplified, and are present in large excess over
PCR
225
so that it can be replicated. This is accomplished by heating the

DNA to near boiling temperatures. In the next step of the cycle,
the solution is cooled to a temperature that favors complementary
DNA sequences finding each other. Since the primers are present
in great excess over the template, they can readily find and basepair with the complementary sequences in the template on either
side of the target sequence. In the third step in the cycle, the DNA
polymerase (which has not been denatured during the heat
treatment because it is thermostable) extends the primer on each
strand, making copies of both DNA strands and doubling the
amount of the target sequence. The cycle is then repeated,
usually about 30 times. At the end of the process, there is a
theoretical yield of 230 more of the target DNA than there was in
the beginning.
Lac Z Blue-White Screening

A powerful tool for biotechnologists is the lac Z gene. You may
recall from an earlier section on the control of gene expression,
that lac Z is part of the lac operon of E. coli and encodes the
enzyme galactosidase. This enzyme catalyzes the hydrolysis of
lactose into glucose and galactose, allowing the bacteria to use
lactose as an energy source. galactosidase can also break
Blue-white Screening Strategy
the template. The DNA polymerase used is chosen to be heat

stable, for reasons that will be clear shortly. The first step of each
PCR cycle involves separating the strands of the template DNA
down an artificial substrate called X-gal to produce a compound

that is blue in color. X-gal can thus be used to test for the
presence of active galactosidase. With this background, we can
now look at how the lac Z gene can be of help to molecular
226
biologists when they create recombinant plasmids. In the
recircularized plasmids having no inserted hGH gene will make
example described earlier, the gene for human growth hormone
functional galactosidase, so in the presence of X-Gal and IPTG
(hGH) was inserted into a plasmid. As we noted, the plasmid, as
these colonies will produce a blue color. This is summarized in
well as the hGH gene are cut with restriction endonucleases to
the figure on the previous page.
create compatible DNA ends that can be ligated. While the ends
of the hGH gene are, indeed, capable of being ligated to the ends
Reverse Transcription
of the plasmid, the two ends of the plasmid could also readily
According to the central dogma, DNA codes for mRNA, which
rejoin. In fact, given that the two ends of the plasmid are are on
codes for protein. An exception to this rule is seen with the
the same molecule, the chances of their finding each other are
retroviruses, RNA-encoded viruses that have a phase in their
much higher than of a plasmid end finding an hGH gene. This
replication cycle during which their genomic RNA is copied into
would mean that many of the ligated molecules would not be
DNA by a virally-encoded enzyme known as reverse
recombinants, but simply recircularized plasmids. Five percent of
transcriptase. The ability to convert RNA to DNA can be useful in
the plasmids having inserts of the hGH gene would be very good.
the laboratory. For example, the power of PCR can be brought to
That would mean that 95% of the bacterial colonies arising from
RNA by converting RNAs of interest to DNA and then amplifying
transformation would contain the original plasmid rather than the
them by PCR. With reverse transcriptase, this is readily
recombinant. To make the process of screening for the relatively
accomplished. First, one creates a DNA oligonucleotide to serve
rare recombinants simpler, plasmids have been engineered that
as a primer for reverse transcriptase to use on a target RNA. The
carry the lac Z gene, modified to contain, with the coding
primer must, of course, be complementary to a segment (near the
sequence, restriction enzyme recognition sites. If one of these
3 end) of the RNA to be amplified. The RNA template, reverse
sites is used to cut open the plasmid and a gene of interest is
transcriptase, the primer, and four dNTPs are mixed. With one
inserted, this disrupts the lac Z gene. If the plasmid simply
round of replication, the RNA is converted to a single strand of
recircularizes, the lac Z gene will be intact. To find which bacterial
DNA, which can be separated from the RNA either by heating or
colonies carry the recombinant plasmids, X-Gal is provided in the
by the use of an RNase to digest the RNA. The product of this
plates. Bacterial colonies containing plasmids with the lac z
process is called a complementary DNA (cDNA).
sequence disrupted by an inserted gene will not produce

functional galactosidase. The X-Gal will not be broken down
and there will be no blue color. By contrast, bacterial cells with
227
Chapter 10
Putting It All
Together
With this chapter, we tie up a bunch of loose ends
and ponder what lies in the future of biochemistry
Putting Everything Together

Looking Back
The bounds of biochemistry have expanded enormously since its inception.
Whlers demonstration, in 1828, that urea could be synthesized outside of a living
Organic chemistry is the study of

carbon compounds. Biochemistry is
the study of carbon compounds that
crawl
cell, showed that there was no vital force that distinguished the chemistry of life
from that of the non-living world. Chemistry is chemistry, but the term
biochemistry was coined in 1903 by Carl Neuberg to describe the special subset
of chemical reactions that happen in living cells. This specialness derives not from
any exceptions to the laws of physics and chemistry, but from the way in which
-Mike Adam.
the chemical reactions in cells are organized and regulated, and also from the
complexity and size of biological molecules.
Faced with far greater complexity than in the inorganic world, the traditional
strategy of biochemists has been divide and conquer. In this approach,
individual enzymes and other biological molecules are purified from cells so that
their properties can be studied in isolation. The underlying logic of this method,
sometimes described as reductionist, is that we can learn about the whole by
studying its individual parts. This painstaking approach, used through most of the
twentieth century, teased out chemical reactions and molecular interactions that
occur within cells, one by one, gradually revealing to scientists much of what we
know in biochemistry today.
229
Biochem is Beautiful
To the tune of Everything is Beautiful
Students study molecules with
All of the structures they possess
Proteins, fats and DNAs
There must be a million ways
To evaluate our knowledge for the test
Biochem is beautiful
Our professor says
From the sugar in our cells
To actions of HDLs
And molecules are dutiful

In every way
Substrates for the enzymes are
Converted e-ver-y day
There is no enzyme
That can lower Delta G
They just work all the time
On transition energy
Catalysis provides to cells

Metabolic jump-startin
They all capitalize
By giving rise
To reactions tween the carbons
Saying it with zest
Would be so much easier
If I could just ace the test
Saying it with zest
Would be so much easier
If I could just ace the test
As increasing numbers of biochemical reactions were worked out,

biochemists began to see that they were connected together in
chains of reactions that we now refer to as metabolic pathways.
These metabolic pathways turned out to be remarkably similar
between cells across all kingdoms of life. Though there are a few
pathways that are unique to certain organisms, many more are
the same, or very similar, in organisms as dierent as bacteria and
humans.
It also became clear that metabolic pathways interacted with
each other via common intermediates or by regulation of one
pathway by molecule(s) created by other pathway(s). The
similarity of the chemical reactions in all living cells was shown to
extend to the common energy currency, ATP, that cells use to
power their chemical reactions, as well as the mechanism by
which cells make the ATP.
Metabolic pathways trace the transformation of molecules in a
cell and represent the work of enzymes, which are proteins. The
discovery of the structure of DNA led to understanding of how
information in genes was used to direct the synthesis of these
proteins. The protein-DNA interactions that determine which
genes are copied into RNA at any given time were uncovered and
helped explain how cells with the same DNA came to express
dierent proteins. The genetic code, as well as the mechanisms
of transcription, translation and regulation of gene expression

also turned out to be remarkably similar in cells of all kinds,
230
leading Nobel laureate Jacob Monod to joke that what was true
Prize was awarded in 1946 for this discovery. Since that time, the
for E.coli was also true for E.lephant.
methods of biochemistry have uncovered all of the information

that you can find in any standard biochemistry textbook, and
The one component at a time approach also helped
more.
biochemists understand how cells

sense changes in their environment
and respond to them. The ability to
sense conditions outside the environs
of cells extends through all groups of
Thousands of enzymes and their substrates have
We are only now beginning to

acquire reliable material for welding
organisms. Even the simplest single-
together the sum total of all that is
celled organism can follow nutrient
known into a whole
gradients to move itself closer to
been identified, and hundreds of metabolic

pathways traced. The structure of hundreds of
proteins is known down to the position of every
atom. Following the elucidation of the structure
of DNA in 1953, scientists have discovered a
- Erwin Schrodinger, 1944 dizzying number of facts about how information is
food. Cells in multicellular organisms
stored, used and inherited in cells. Cloned and
can detect chemical cues in the
transgenic animals and gene therapy were a
blood (nutrients, hormones) or impulses from nerve cells and alter
reality in less than 50 years. And the discoveries still keep
their actions. These cues may trigger changes in metabolism,
coming.
decisions to divide, die, or become senescent, or the

performance of specialized functions (e.g., muscle contraction or
Looking Forward
enzyme secretion). Thus cells are constantly in a state of flux,
But toward the end of the twentieth century, new methods began
adjusting their activities in response to signals from outside
to change the face of biochemistry. The launching of the Human
themselves as well as their own changing needs.
Genome Project and the development of faster and cheaper
The power of the take things apart analytical approach is

evident from the astounding pace of discoveries in biochemistry
and molecular biology. The first demonstration that an enzyme
was a protein was made only in 1926, and it wasnt till twenty
years later that this was sucient well established that the Nobel
sequencing technologies provided biochemists with entire

genome sequences, not only of humans, but of numerous other
organisms. Huge databases were set up to deal with the volume
of sequence information generated by the various genome
projects. Computer programs cataloged and analyzed these
sequences, making sense of the enormous quantities of data.
231
Thank God There's a Video
To the tune of "Thank God I'm a Country Boy"

Students sing text in RED
There's a bundle of things a student oughta know
And Ahern's talk isn't really very slow
Learnin' ain't easy / the lectures kinda blow
Thank God there's a video
Well weve gone through the cycles and their enzymes too
Studying the regulation everything is new
I gotta admit that I havent got a clue
What am I gonna do?
So I got me a note card and bought me a Stryer
Got the enzymes down and the names he requires
I hope that I can muster up a little more desire
Just got up to speed about the NAD
Protons moving through Complex Vee
Electrons dance in the cytochrome C
Gotta hear the MP3
Fatty acid oxidation makes the acetyl-CoA

Inside the inner matrix of the mitochondri-ay
It's very complicated, I guess I gotta say
I hope that I can muster up a little more desire
Replication's kind of easy in a simple kind of way
Copyin' the bases in the plasmid DNAs
Gs goes with Cs and Ts go with As
Thanks to polymerase
And the DNA's a template for the RNA
Helices unwinding at T-A-T-A
Termination happens, then the enzyme goes away
Don't forget the poly-A
I think that I can muster up a little more desire

232
Protein coding regions of genomes could be identified and
among others. As an example, let us consider proteomics. The
translated in silico to deduce the amino acid sequence of the
field of proteomics is concerned with all of the proteins of a cell.
encoded polypeptides. Comparisons could be made between
Since proteins are the workhorses of cells, knowing which ones
the gene sequences of dierent organisms. In parallel with the
are being made at any given time provides us with an overview of
growth of sequence information, more and more protein
everything that is happening in the cells under specific conditions.
structures were determined, by using
How is such an analysis
X-ray crystallography and NMR

spectroscopy. These structures, too,
It is an old saying, abundantly justified, that where
were deposited in databases to be
sciences meet there growth occurs. It is true moreover to
accessible to all scientists.

The accumulation of vast amounts of
sequence and structure information
went hand in hand with new and
performed? First, one

extracts all of the
proteins from a given cell
say that in scientific borderlands not only are facts
type (liver, for example).
gathered that [are] often new in kind, but it is in these
Next, the proteins are
regions that wholly new concepts arise.
ambitious goals for biochemistry.
separated in a two-step
gel method, where the
Sir Frederick Gowland Hopkins first step resolves
Modern biotechnology techniques
proteins based on their
have provided tools for studying
charge and the second
biochemistry in entirely new ways. The old ways of dividing and
separates them by mass. The product of this analysis is a single
conquering to study individual reactions are now being
gel (called a 2-D gel) on which all of the proteins have been
supplemented by approaches that permit researchers to study
separated. In the left-right orientation, they dier in their original
cellular biochemistry in its entirety.
charge and in the up/down orientation, they dier in their size.
These fields of research, which collectively are often referred to as
By using such a technique, as many as 6000 cellular proteins can
the -omics include genomics (study of all the DNA of a cell),
be separated and visualized as spots on a single gel. Robotic
proteomics (study of all the proteins of a cell), transcriptomics
techniques allow excision of individual spots and analysis on
(study of all the transciption products of a cell), and
mass spectrometers to identify every protein present in the
metabolomics (study of all the metabolic reactions of a cell),
original extract.
233
Why is this useful? There are several ways in which this
carbohydrates, etc., are allowing biochemists to have, for the first
information can be illuminating. For example, by comparing the
time, a big picture view of the activities of cells. While these
proteins in a normal liver cell with those in a cancerous liver cell,
techniques have already provided valuable new insights, they are
one can quickly determine if there are any proteins that are
still incomplete, as a description of what goes on in cells. This is
expressed or missing only in the cancer cells. These dierences
because they provide us with a snapshot that captures what is
between normal and cancerous cells may provide clues to the
happening in cells at the moment that they were disrupted to
mechanisms by which the cancer arose or suggest ways to treat
make the extract. But cells are not static entities. At every
the cancer. Or, the same sort of analysis could be done on cells
moment, they are adapting their activities in response to changing
to find out about the eects of a hormone

or drug treatment. Comparison of the
proteins found in untreated and treated
cells would give a global view of the
combinations of internal and external
Almost all aspects of life are engineered conditions. Changes in response to any
at the molecular level, and without
one signal are modified and influenced by

the every other condition, within and
protein changes resulting from the
understanding molecules we can only
treatment.
have a very sketchy understanding of
complex systems as an integrated whole is
life itself.
the new holy grail of biochemistry.
Similar analyses can be performed on
outside the cell, and understand these
the mRNA of cells, employing devices
Francis Crick The aim, then, is to develop models that
called microarrays. In this case, all the
depict these dynamic interactions within
RNAs that are being made at the time that the cell extract is made
cells, and to understand how such interactions give rise to the
can be identified by the signals generated when the RNAs
properties and behavior that we observe. This is the goal of the
hybridize with oligonucleotides complementary to their sequence,
emerging field of systems biology that constructs mathematical
that are immobilized in ordered arrays on the surface of a plate.
models and simulations, based on the large data sets generated
The position and strength of these signals indicates which RNAs
by transcriptomic, proteomic and other broad-range techniques.
are made and in what amounts.
Systems biology is truly an interdisciplinary venture, drawing as it
The techniques of proteomics and transcriptomics, together with

other global view approaches of molecules like lipids,
does on mathematics and computer science as much as

traditional bench biochemistry. While the original laboratory
techniques of biochemistry are by no means obsolete, they will
234
no longer be the sole tools used to
oxidation damage. This idea was
understand what goes on inside of

cells.
tested by screening large numbers of
To the tune of Winter Wonderland
compounds for the ability to inhibit a
Milam Hall - Its 12:30

And Aherns gettin wordy
He walks to and fro

While not talkin slow
Givin it to B-B-4-5-0
I was happy when the term got started

Lecture notes and videos galore
MP3s got added to my iPod
But recitations sometimes were a bore
And exams bit me roughly

When the curve turned out ugly
I dont think its so

My scores are too low
Slidin by in B-B-4-5-0
Final-LY theres an examination

On December 9th at 6:00 pm
Ill have my card packed with information
So I dont have to memorize it then
And Ill feel like a smarty

With my jam-packed note-cardy
Just one more to go
And then ho-ho-ho
Ill be done with B-B-4-5-0
These newer approaches are already

leading to applications that are of
tremendous value. Understanding the
system level dierences between
normal and diseased cells can lead to
major changes in the way diseases are
detected, treated or altogether
prevented.
One recent triumph of systems biology
has been in an intriguing discovery
about how antibiotic drugs work.
System level studies of many classes
of antibiotics revealed that, regardless
of how we think they work to kill
bacteria, all of the drugs appear to
have a common eect that of
increasing the level of oxidative
damage, leading to cell death. This
BB Wonderland
their oxidation-damaged DNA. This

screen yielded several compounds, the
best of which was able to increase the
eectiveness of the drug gentamicin by
about a thousand-fold. Such
compounds will be of increasing value
in a world where antibiotic resistance is
on the rise.
Another application of systems biology
is in the development of more eective
vaccines. Till recently, most vaccines
have been developed with little
understanding of how exactly they
stimulate the immune response. As
systems biology approaches give us a
better understanding of the changes
that vaccines bring about to mediate
immunity, it will be possible to identify
observation suggested that the
the patterns that characterize stronger
potency of antibiotics could be
immune responses or adverse
enhanced by blocking bacterial

responses that protect against
pathway that bacteria use to repair

reactions to vaccines and even to

predict how well particular vaccines
235
The tert alcohol was a fool

Turned to ketone in making a fuel
The work is complete
The bond gave up heat
As it lost all its family joules
may work in specific
programmed to make biofuels that could potentially replace
populations or individuals.
petroleum.
Similarly, system level

studies can help identify
which drugs might be most
eective, with the fewest side-eects, for a given patient, leading

to a new era of personalized medicine.
The successes of systems and synthetic biology, even in their

infancy, promise great advances both in our understanding of
living systems and in the applications that arise out of that
knowledge. The next fifty years in biological research may well
eclipse even the amazing accomplishments of the last. The
Related to systems biology, and heavily dependent on it, is
practice of medicine will be transformed. Regenerative medicine
synthetic biology, which aims to use the knowledge gained from
will improve, as a better knowledge of stem cells allows us to use
the former to engineer novel biological systems and pathways.
them more eectively to replace cardiac muscle lost in a heart
Because the technology now exists to

synthesize extremely long pieces of DNA,
entire genomes can be made synthetically
and used to program cells that they are
inserted into. It also allows for the possibility
The drivers ed student gave holler

For a driving course worth every dollar
When she passed it today
Her friends had to say
It made her some kind of road scholar
of custom-designing an organism to create
attack, neurons damaged in Parkinsons or

Alzheimers, or even to regrow limbs lost in
accidents or war. Treatments for our illnesses
can be tailored to be optimal for each
individual. Biofuels may bail us out when oil
supplies run out and engineered organisms
particular chemical compounds through artificially assembled
may help clean up a polluted planet. And research on longevity
pathways.
may give us the best gift of all- lives extended long enough to
These methods have already been used to produce the drug

artemisinin, which is used to treat malaria. The pathway for
making a precursor of artemisinin was created by combining a
metabolic pathway from yeast with part of another derived from
the plant Artemisia annua, the natural source of artemisinin.
Similar eorts are underway for anticancer drugs, novel drugs,
flavoring compounds, etc. One major goal is to create organisms
witness these advances and to participate in the creation of a

new and better world.
The science researcher would whine
At his data darn near every time
When it comes to a graph
Theres no cause to laugh
He wonders where he should draw the line
236
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
237
Chapter 11
Outlines,
Notes,
Exams
In this section we include material from the classes
we teach at Oregon State University. This includes
notes, outlines, lecture videos, reviews, and exams
we have given to our students
Appendix - Class Materials

APPENDIX 1
This section of the appendix includes links to
materials we use to teach our classes. In
each case, the hyperlinks to the Outline
material and the Videos will take you to an
outside Web site, so you will thus need an
Internet connection to those materials. The
Key Point Summary links lead to relevant
sections in Appendix 2.
Appendix I - Class Materials

This section is organized dierently from the rest of the text. It is the e-book
equivalent of a hybrid Internet/ebook-based appendix. In this section, we place an
organized collection of links to videos, online outlines of hyperlinks, and note
summaries for the many topics in the first nine chapters.
From BB 450/550
Introduction to Biochemistry / Acids, Bases, Henderson Hasselbalch

Key Points Summary HERE / HERE

Video HERE / HERE /
Protein Structure

Key Points Summary HERE

Video HERE / HERE /HERE
Protein Characterization


Video HERE / HERE / HERE
Hemoglobin


Video HERE / HERE
Enzymes


Video HERE / HERE / HERE / HERE
Catalytic Strategies


239
Allostery and Regulation


Carbohydrate Structures


Cellular Signaling


Metabolic Control


Video HERE / HERE
Glycolysis & Gluconeogenesis


Glycogen Metabolism


Review Sessions

From BB 451/551
Citric Acid Cycle


Video HERE / HERE
Lipids and Membranes


Video HERE / HERE
Membrane Transport


Video HERE / HERE
Mitochondria / Electron Transport


Photosynthesis

Steroid and Lipid Metabolism


Video HERE / HERE
Fatty Acid and Prostaglandin
Metabolism




Video HERE / HERE
Nitrogen Metabolism

DNA Replication, Recombination,
Repair


Transcription


Translation


Gene Regulation


Sensory Systems


Review Sessions

From BB 350
Introduction

Video HERE
Water/Biochemistry

Video HERE / HERE
Amino Acids/Peptides

Video HERE
Protein Structure

Protein Purification

Video HERE / HERE
Enzymes

Control of Enzymes

Membranes

Nucleic Acids

Video HERE / HERE
DNA Synthesis

RNA Synthesis

Protein Synthesis

Biotechnology
240
Video HERE / HERE
Viruses and Cancer

Other YouTube Video

Collections
Video HERE / HERE
Metabolic Energy
BB 350 (older - 139 videos) HERE
BB 350 (newer - 36 videos) HERE
Video HERE / HERE
Carbohydrates
Bite Sized Biochemistry (older - 53
videos from BB 450/451) HERE
Video HERE / HERE
Glycolysis
Comprehensive YouTube Collection
(375+ videos) HERE
Gluconeogenesis and Glycogen

Citric Acid Cycle

Video HERE / HERE
Kevin also teaches a biochemistry course (BB

100) that has no pre-requisites and is aimed at
anyone interested in learning about the subject
without technical details. It can be found HERE
Electron Transport /
Oxidative Phosphorylation

Video HERE / HERE
Lipid Metabolism

Photosynthesis

Video HERE
Nitrogen Metabolism

Video HERE / HERE
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
241
Section 2
Appendix - Key Points

APPENDIX 2
Key Points - Basics
In this section, we include all of the key points

of our lectures. Hyperlinks to all of the topics
in this section are provided in Appendix 1.
1. Covalent bonds are VERY strong bonds that hold atoms/molecules together.
Covalent bonds are the 'glue' that holds together biomolecules.
2. Hydrogen bonds are much weaker bonds that are also important in biological
molecules. Hydrogen bonds arise from uneven sharing of electrons between, for
example a nitrogen and a hydrogen or an oxygen and a hydrogen. In each case,
the hydrogen ends up with a partial positive charge and the other atom has a
partial negative charge. The partial positive charge of the hydrogen may be
attracted to a partial negative charge on another oxygen or hydrogen. These
bonds are weaker than covalent bonds, but are VERY important in stabilizing
protein and DNA structures.
3. Water has its relatively high boiling point due to its numerous hydrogen bonds.
The double helix of DNA is held together by hydrogen bonds between the
individual bases.
4. Hydrogen bonds are additive, so they provide great stability in numbers (as in
across a chromosome), but
The mouse in my house makes me shriek
less stability locally (one can
Every time I espy its physique
easily pull them apart when
The thing that inflames
DNA needs to replicate).
Me most is his games
5. Hydrogen bonds are some
He likes to play hide and go squeak
of the stabilizing forces of
proteins. Since protein
242
function depends on protein structure, forces that disrupt

hydrogen bonds (such as heat) tend to disrupt protein structure
and function. It is because of this that cooking food kills bacteria,
because it denatures their proteins. Since proteins are the
"workhorses" of cells, loss of protein function means loss of cell
function, which means death.
6. pH is a measure of the proton concentration in a solution. The
pH is the negative log of the hydrogen ion concentration. The
lower the pH, the higher the hydrogen ion concentration and the
stronger the acid. pH + pOH = 14. The pOH is the negative log of
the hydroxide ion concentration. The pKa is, therefore, the
negative log of the Ka. The lower the pKa for an acid system (such
as the acetic acid system discussed in class), the stronger the
acid is.
7. Acetic acid (HAc above) is a weak acid, meaning that it doesn't
completely dissolve in water. HCl, by contrast is called a strong
acid because it completely dissociates in water.
HAc <=> H+ + AcWe can write the general equation for the dissociation of ANY
weak acid as
HA <=> H+ + A-
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
243
4. pKa is a measure of the

strength of an acid. The lower
the pKa, the stronger the acid.
Key Points - Buers

1. Weak acids (written as HA)
dissociate in water to a limited
extent. This ionization can be
written as
5. Very strong acids like HCl that

completely ionize in water do
not have pKa values.
HA <=> H+ + A-
6. Buers are systems of

molecules in solution that act to
resist pH changes within
approximately 1 pH range of
their respective pKa values. For
example, acetic acid has a pKa
of 4.76. This means it will be an
eective buer in the range of
3.76 to 5.76.
(where <=> refers to the

equiilibrium symbol)
2. For dissociation of such weak
acids, the HendersonHasselbalch equation applies. It
states that
pH = pKa + log {[A-]/[HA]}
where the bracketed item (such
as [A-]) refers to the
concentration of that item.
7. At the pKa of a buer, it will

have equal amounts of
molecules with the appropriate
proton and missing the proton.
For acetic acid, for example,
(abbreviated as HAc ), at pH
4.76, it will have equal amounts
of HAc and Ac-. Below pH 4.76,
it will have more HAc than Ac-.
Above pH 4.76, it will have more
Ac- than HAC.
3. Remember that pH is defined

as the negative log of the
hydrogen ion concentration
-log[H+]
and the pKa is defined as the

negative log of the acid
dissociation constant, Ka
-log(Ka)
8. When referring to a buer

system, we shall use the term
'salt' to refer to the molecule of
244
the buer system that has lost a proton.

We shall use the term 'acid' to refer to the
molecule of the buer system that has not
lost its proton. The dierence between a
salt and an acid is a single proton.
The corn chippers did not think it neato

When the famous judge came incognito
And marked all their Lays
With a bunch of O.J.s
So that was why Frito banned Ito
9. Adding a strong acid (such as HCl) to a

buer system causes salt (A-) to be converted into an acid (HA).
For every molecule of HCl added, one A- is converted to one
HA. Thus, if one had 500 molecules of HA and 500 molecules
of A- in solution and one added 10 molecules of HCl, the
resulting solution would have 510 molecules of HA and 490
molecules of A-.
10. Conversely, adding a strong base (such as NaOH) to a buer
system causes HA to be converted to A-. For every molecule of
NaOH added, one HA is converted to one A-. Thus, if one had
500 molecules of HA and 500 molecules of A- in solution and
added 10 molecules of NaOH, the resulting solution would
have 490 molecules of HA and 510 molecules of A-.
11. Molecules can have more than one buering region. Alanine,
for example has both an amine group and a carboxyl group
that can gain/lose protons (hydrogen ions). It will thus have
two pKa, one for the carboxyl group and one for the amine
group.
12. A buer system will be at maximum
capacity when the concentration of the
undissociated acid (HA) equals that of
the salt (A-). That is (Acid = Salt). The
Henderson Hasselbalch equation further
reveals that when this is true, pH = pKa.
13. Amine systems (also in amino

acids) have two forms:
NH3+ and NH2.
Note that the NH3+ is the acid and NH2
is the salt in our nomenclature.
14. Carboxyl systems have two forms too. COOH has no charge
and when it loses its proton, COO- has a negative one charge.
15. Students make mistakes calling things acids and bases based
solely on structure. Consequently, they get confused when
they think of an amine like NH3+ donating protons (acting like
an acid). The same students usually aren't confused, however,
about COO- accepting protons (acting like a base). That is
why we avoid calling amine groups "bases" here.
16. The Henderson Hasselbalch equation tells us we can predict
the ratio of salt to acid as a function of pH if we know the pKa.
Consequently, we can predict the charge on amino acids in a
protein as the pH changes. Subtle changes in pH in the body
can have drastic changes in protein structure and function.
For example, hemoglobin undergoes drastic changes
between the lungs (relatively high pH) and actively respiring
tissue (relatively low pH). The pH changes are not very large,
so it is important to recognize that very small pH changes can
make a big dierence.
When the hospital beds of last quarter

Didnt fill up the income was shorter
The medical center
Considered some renters
But remained just as doctors without boarders
17.The value of the Henderson

Hasselbalch equation is that by
knowing the pH and the pKa of a
molecule, the approximate charge of it
in solution can be determined. For this
245
book, we will assume that if the pH is more than one unit

below the pKa of a group, the proton is ON it. If the pH is
more than one unit above the pKa of the group, the proton is
OFF it. This is only useful for estimating charge. Determination
of the pI (pH at which a molecule has a charge of exactly zero)
requires calculation.
The iPhone support sta saidWe
Make software thats alwaysbug-free
I replied to the jerk
That it just wouldnt work
Cuz it was an app errant tome
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246
Key Points - Protein

Structure
1. Protein structure dictates protein
function. The structure of a protein
is a function of the sequence of
amino acids comprising it.
2. Amino acids are the monomeric
(building block) units of proteins.
They are covalently joined together
in peptide bonds to make proteins
(polypeptides).
3. There are 20 amino acids commonly
found in proteins and of these 20,
19 have a chiral center and thus can
exist in two stereoisomeric forms.
The only one that doesn't have a
chiral center is glycine. Almost all
biologically made amino acids are in
the same stereoisomeric form - the
'L' form. The 'D' form occurs only in
very rare peptides, such as in the
cell wall of bacteria (discussed later
in the book).
4. Amino acids are grouped into
several structural categories based
on the composition of their R

groups. All biochemistry students
should know the names of the 20 amino acids of proteins,
which of the groups above each one

belongs to and they will need to be
able to predict ionization at given pH
values if you are supplied pKa values.
Of the 20 amino acids in proteins, the
ones we will be concerned with have
R groups that can ionize. These
include the aminos, the carboxyls,
the sulfhydryl (cysteine) and the
hydroxyl of tyrosine.
5. I mentioned briefly some of the
properties of individual amino acids
in the lecture. You should know those
general properties (such as
hydrophobic, hydrophilic, sulfhydryl,
and ionizing side chains.) Glycine is
the simplest amino acid because it
only has a hydrogen as its R-group. It
is the only amino acid that doesn't
have D and L forms. Another
important one structurally is proline.
6. Molecules, such as amino acids,
that are capable of gaining/losing
more than one proton, have pKa's
corresponding to each functional
group that can lose a proton. For
example, a simple amino acid, such
as alanine, has an amino group (pKa
about 9.5) and a carboxyl group (pKa about 2.5).
247
7. A titration plot for a simple amino acid (no R

groups that can lose protons) has two
flattened regions - each one occurring at
the respective pKa. See the problem solving
video HERE for more information.
At the North Pole an elf-tracking thing

Uses letters but no numbering
Elf A may be fast
And Elf Z is the last
But Elf S is forever the king
8. A zwitterion is a molecule whose net charge

is zero.
9. The pI of a molecule is the point at which its charge is exactly
zero. The assumptions above about protons on/o cannot be
used to find the pI. It must be calculated. The pI of a molecule
is the average of the pKa values around the point on the
titration curve where the charge is zero
10. The sequence of amino acids in a protein is ultimately
responsible for all of the properties a protein has. The
sequence of amino acids of a protein is referred to as its
primary structure.
ratio.
13.The bonds around the alpha carbon

(the carbon bonded to both an amino
group and a carboxy group) can both
rotate, however, because they are single
bonds. One can thus describe a
polypeptide as a series of planes separated by an alpha
carbon, with the planes each being rotated a certain number
of degrees relative to the alpha carbon. If we think of the
alpha carbon as being in between two planes, then the plane
on the left can rotate (theoretically) 360 degrees and the plane
on the right can also theoretically rotate 360 degrees. These
angles of rotation of planes are referred to as phi and psi
angles. Phi refers to the rotational angle around the single
bond between the alpha amino group and the alpha carbon.
Psi refers to the rotational angle around the single bond
between the alpha carbon and the alpha carboxyl group.
11. Bonds holding amino acids together in a protein are called

peptide bonds and they occur between the alpha amino
group of one amino acid and the alpha carboxyl group of the
next one.
14. In the early 1960s, G. N. Ramachandran, C. Ramakrishnan,

and V. Sasisekharan recognized that not all rotations of phi
and psi would be theoretically feasible because steric
hindrance would preclude some rotational positions. They
plotted theoretical rotations of psi versus phi and calculated
12. Peptide bonds form resonant structures such that the bond
which of these angles would provide stable structures. The
itself behaves like a double bond. Double bonds cannot rotate
resulting plot came to be known as a Ramachandran plot. In
(unlike single bonds) and thus they define a plane. Alpha
it, the regions of predicted stability turn
carbons on either side of a peptide bond are
In the shuttle it seems to confound
out to be regions of known stability from
generally arranged in a trans configuration
The astronauts high oer the ground
protein structures that have been
(about 10,000 trans to one cis), except when
Every time when they read
determined.
proline is involved. Peptide bonds involving
It's almost guaranteed
proline favor the trans by about a 100 to 1
15.The secondary structure of a
They can't ever put their books down
248
polypeptide refers to regular/repeating

structure(s) arising from interactions
between amino acids that are
relatively close together in primary
sequence. This means less than 10
amino acids away.
In St. Louis the plannerscould see

That their Gateway might notcome to be
The people opposing
The structure imposing
Saw it as their arch enemy
16. One protein secondary structure that is stable in both real

proteins and theoretical ones (Ramachandran plots) is the
alpha helix. Alpha helices are one type of secondary structure
and form coils.
17. Hydrogen bonds are primary forces stabilizing secondary
structures. In alpha helices, carbonyl oxygen from a peptide
bond forms a hydrogen bond with an amine nitrogen of
another peptide bond four amino acids distant.
18. Certain amino acids with simple side chains, such as alanine,
are very favorable for formation of alpha helices, whereas
bulky (tryptophan) or cyclic (proline) amino acids tend to
disrupt alpha helices. Thus, one can reasonably accurately
predict from an amino acid sequence which regions of a
protein sequence will exist as alpha helices and which will not.
and others (proline again) favor its

disruption.
20.Beta sheets arise from arrangement of

beta strands. These arise from
interactions (hydrogen bonds) between
beta strands (parallel or antiparallel), such
that the carbonyl oxygen of one side interacts with the amine
hydrogen with the other. Beta strands can also orient their R
groups such that they interact appropriately (hydrophobichydrophobic, for example). Beta sheets can be oriented in
several ways.
21. Essential features of proteins that are essential for their overall
structure are turns. Turns interrupt secondary structure (alpha
helices and beta strands) and often involve proline and glycine
residues.
22. Another type of fibrous protein is collagen, the most abundant
protein in your body. It contains three intertwined helices
comprised of abundant repeating units of glycine, proline, and
hydroxylproline
23. Hydroxylation of proline is a post-translational modification
(occurs after the protein is made) and the hydroxyls are
placed there in a reaction that uses vitamin C.
19. Another type of common secondary structure commonly

found in protein is the beta strand (note that the term beta
sheet refers to layering together of beta
strands together), which consists of amino
Out at State Farm the agent extends
acid backbones in a V shape (like the pleats
A payment to tie up loose ends
of a drape). A beta strand can be thought of
This brings to a close
as a helix in two dimensions though that is
What the owner now knows
The way that a Mercedes bends
an over-simplification. Again, specific amino
acids favor the formation of this structure
24.The hydroxyls of hydroxyproline can

react with other, forming covalent crosslinks that make the collagen fibers more
sturdy.
25.Tertiary structure relates to interactions
between amino acids in a protein that are
249
not close in
primary sequence.
These interactions
are made possible
by folding of the
protein chain to
bring the distant amino acids closer together.
When at bedtime the kid was a pest
Mom and Dad only had to suggest
If he just wouldnt stop
They would go get a cop
And then charge with resisting a rest
26. Tertiary structure is stabilized by disulfide bonds, ionic

interactions, hydrogen bonds, hydrophilic, and hydrophobic
interactions. Disulfide bonds are the strongest forces holding
tertiary structure together, as they are covalent bonds.
27. Most proteins that are in cells are globular in nature.
28. Myoglobin is a protein that acts as an oxygen 'battery',
storing oxygen in muscles for when it is needed. Myoglobin
contains a heme group that contains iron. Heme is a
'prosthetic group', which refers to a non-amino acid
containing group that binds to a protein and augments its
function.
29. Amino acid residues in myoglobin are arranged such that
hydrophilic amino acids are arranged on the outside and
hydrophobic amino acids are largely arranged on the inside.
30. Porin is a membrane protein. Proteins embedded in
membranes often have external amino acids that are
hydrophobic so they can interact with the non-polar portions
of membranes. Porin, in addition, has a hole in the center that
allows water to pass through it. The amino acids in porin are
arranged with non-polar amino acids outside and polar amino
acids inside.
31. Quaternary structure of proteins relates to the interactions

between separate polypeptide chains within the protein. The
word polypeptide refers to a polymer of amino acids. A
protein may contain one or more polypeptides, is folded and
may be covalently modified.
32. Hemoglobin (and many other proteins) have multiple
polypeptide subunits. Interactions between the subunits
include disulfide bonds, ionic interactions, hydrogen bonds,
hydrophilic, and hydrophobic interactions. Modification of the
quaternary structure of a protein may have the same eects
as modification of its tertiary structure - alteration of its
function/activity.
33. Folding is necessary for proteins to assume their proper
shape and function. The instructions for folding are all
contained in the sequence of amino acids, but we do not yet
understand how those instructions are carried out rapidly and
eciently. Levinthal's paradox illustrates the fact that folding
is not a random event, but rather based on an ordered
sequence of events arising from the chemistry of each group.
34. Proper folding of a protein is essential. Cells have complexes
called chaperonins that help some proteins to fold properly.
Misfolding of proteins is implicated in diseases, such as mad
cow disease and Creutzfeld-Jacob disease in humans. The
At the dairy, Ms Margo R. said
Looking up where they make the cheese spread
I dont get the jokes
Of these curd forming folks
I guess its whey over my head
250
protein may have the same eects as modification of

its tertiary structure - alteration of its function/
activity.
36.The enzyme ribonuclease (RNase) is interesting in
being very stable to heat and other things that
denature/inactivate other proteins. (By the way,
denaturation is a word that means the tertiary and/or
quaternary structure of a protein is disrupted.).
RNase has disulfide bonds that help it to remain
resistant to denaturation.
37. Some chemicals, such as mercaptoethanol, can
reduce the disulfides (between cysteine residues) in
proteins to sulfhydryls. In the process of transferring
electrons to the cysteines, the sulfhydryls of
mercaptoethanol become converted to disulfides.
Treatment of RNase with mercaptoethanol reduces
RNAse's disulfides to sulfhydryls. Subsequent
treatment of RNase with urea disrupts hydrogen
bonds and allows the protein to be denatured.
causative agent in these diseases is a "contagious" protein

that is coded by the genome of each organism. When it
doesn't fold properly, it helps induce other copies of the same
protein to misfold as well, resulting in plaque-like structures
that destroy nerve cells.
35. Hemoglobin (and many other proteins) have multiple
polypeptide subunits. Interactions between the subunits
include ionic interactions, hydrogen bonds, and hydrophobic
interactions. Modification of the quaternary structure of a
38. Interestingly, removal of the mercaptoethanol and urea from

the solution allows RNase to refold, reestablish the correct
disulfide bonds, and regain activity. Clearly, the primary
sequence of this protein is sucient for it to be able to refold
itself to the proper configuration.
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1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
251
Key Points Macromolecular

Characterization
1. Proteins sometimes have amino
acids in them that are chemically
modified. Chemical modification of
amino acids in proteins almost
always occurs AFTER the protein is
synthesized (also described as
post-translational modification).
Examples include hydroxyproline
and hydroxylysine in collagen,
gamma carboxyglutamate in
prothrombin, and phosphoserine in
many other proteins. Modification of
the collagen residues allows for the
triple helical structure of the protein
and for the strands to be crosslinked (an important structural
consideration).
2. Purification of proteins exploits
dierences in charge, size, shape,
and anity for specific compounds.
Centrifugation (artificial gravity)
provides a means of precipitating
cellular components. The amount of
centrifugal force applied (speed of

spin) determines the rate with which
a given substance will be driven towards the bottom of the
tube. The largest components will

precipitate the fastest at the slowest
speeds. The smallest components will
precipitate the slowest and may
remain in solution, allowing one to
fractionate materials based on size
and solubility in water.
3. Dialysis provides a means of
separating large molecules (like
proteins, DNA, etc.) from small
molecules (like salts, etc.) by encasing
the protein/salt mixture in a
membrane. The membrane has holes
(pores) big enough to let small
molecules to pass through it, but too
small to let out the big molecules.
Thus, with this technique, one can
eectively remove the salt from a
protein.
4. Gel filtration (gel exclusion
chromatography) provides another
way to separate large molecules from
smaller ones. It employs beads with
tunnels in them. The beads are packed
in a column. The beads have buer
running through them and the
openings to the tunnels are a fixed
size (called an exclusion limit). The
size of the opening determines the maximum size of molecule
that can enter it. Molecules smaller than the exclusion limit
252
enter the beads

and travel a
longer path than
molecules larger
than the
exclusion limit.
Thus, when large and small proteins are applied to a column,
the large proteins come through first and the small ones come
last.
A Chinese composer named Ong
Wrote a beautiful lyrical song
Except he did make
A single mistake
That he would never change, writer Ong
5. Ion exchange chromatography uses beads in a column also,

but instead of tunnels, the beads are coated with a molecule
having either a positive or negative charge. If a mixture of
molecules with positive and negative charges is added to the
column, the negative molecules will "stick" to the column when
the beads are coated with positive charges. The molecules with
positive charges will not stick to the positively charged beads
and thus one can separate positive and negative molecules
using a column containing beads with only one type of charge.
6. Anity chromatography exploits the tendency of many proteins
to 'bind' to molecules. For example, many proteins bind ATP. If
one takes beads and coats them with ATP and then passes a
protein mixture through the column, only those proteins that
bind to ATP will stick to the column. The others will pass
through it freely. With anity chromatography, one can
separate proteins that bind a specific molecule from proteins
that don't bind that molecule.
7. HPLC (High Performance Liquid Chromatography) employs
densely packed columns containing material with chemical
groups on them that interact with molecules as they are
pumped through the column. The most popular type of HPLC

is reverse phase chromatography, which uses column material
that is very non-polar. Hydrophobic interactions cause the less
polar molecules to be retained the longest by the column,
whereas the polar molecules have no anity for the column
material and pass through first.
8. Gel electrophoresis is a method for separating molecules using
an electric field across a support (gel). In this method,
molecules separate by size. The smallest ones move the
fastest and the largest ones move the slowest.
9. Isoelectric focusing is a technique that separates molecules on
the basis of their pI (pH at which their net charge is exactly
zero). It is performed in tubes containing special compounds
(polyelectrolytes) that migrate to specific points in the tube
when in the presence of an electric field. This eectively
creates a pH gradient from one end of the tube to the other. If
proteins are added to the tube as the gradient is getting
established, they will migrate to the point in the tube where the
pH corresponds to their pI and they will migrate no further,
since they will have a charge of zero.
10. 2D gel electrophoresis is a powerful tool for proteomics that
combines the techniques of isoelectric focusing with SDSPAGE. In this method, proteins are first separated according to
their pI values by
The octuplets mother and dad
isoelectric focusing.
Reflected on children they had
Then the tube from
Their bundles of joys
the isoelectric
Make ear piercing noise
focusing is applied
So now they are stork raving mad
to the top of an
253
SDS-PAGE gel and the proteins are separated by size. The

result is a two dimensional separation of virtually every protein
in the cell.
11. Nucleic acids (which are negatively charged and 'rod-like' in
shape) can be separated by agarose gel electrophoresis
readily. In this technique, a 'gel' is made that consists of a
matrix material (agarose) that forms a sort of 'mesh' of hole
through which the DNA molecules pass. Electric fields are used
to separate macromolecules by size. The sample is loaded on
the top of the gel and electrical current is passed through it
such that the bottom electrode is positive and the top one is
negative. Negatively charged DNA molecules at the top of the
gel are driven away from the top towards the bottom. Small
molecules make their way through the gel fastest and big
molecules travel more slowly. The key to gel electrophoresis is
to have all of the molecules being separated have a negative
charge.
12. Proteins, however are usually globular in their native state
and, without other modification, may be negatively, positively
OR neutrally charged. Another type of electrophoresis employs
polymers of acrylamide (called polyacrylamide) to form the
mesh of the gel. This mesh has smaller holes that agarose and
allows separation of smaller molecules like proteins. To
separate proteins on the basis of size, the detergent sodium
dodecyl sulfate
Atthe plant singles bar for a while
(SDS) is add to
Theanther developed a smile
the protein
Itwas clear as a crystal
mixture, causing
Headored every pistil
the proteins to
Whenhe told them that I like your style
denature, assume
rod-like shapes,
I dont have a goodmemory
and be coated
Of the things that Ive donerecently
I am still at a loss
uniformly with
For the boomrang Itossed
the negative
Perhaps it will come backto me
charge of the
SDS.
Consequently all proteins in the mixture obtain a negative
charge and can be separated just like DNA on SDSpolyacrylamide gel electrophoresis (SDS-PAGE).
13. During purification of proteins it is important to follow the
purification process. At each step of the process, a small
sample of the protein extract is taken and the total amount of
protein, and the amount of activity of the desired protein are
measured. The specific activity of the protein in the tube is the
amount of activity (in units) divided by the total mass of protein.
The yield of the desired protein at any point in the purification
process is the number of units of the desired protein at that
point divided by the number of units that one started with. The
purification level at any point is the specific activity at that point
divided by the specific activity one started with.
14. Breaking large proteins down into smaller pieces is also
important for studying them. Some reagents include cyanogen
bromide (breaks bonds on carboxyl side of methionine residues
in a protein) and enzymes known as proteases. One such
protease is trypsin, which cleaves on the carboxyl side of lysine
and arginine residues in a polypeptide. Thrombin is another
enzyme that breaks peptide bonds. It cleaves them on the
carboxyl side of arginine in a polypeptide.
254
15. Immunological techniques aid in

identifying specific proteins.
Antibodies (immune system
proteins) recognize and bind to
specific structures. The structures
antibodies bind to are called
antigens. Usually antibodies are
targeted against specific protein
structures. Since proteins dier from
each other in their structure,
molecules (like antibodies) that bind
to specific structures will bind to
specific proteins.
16. Antibodies are very useful in that
they can be linked to fluorescent
dyes, gold particles, or enzymes to
help one visualize where an
antibody has bound. They are useful
in binding to specific cellular
structures (in situ hydridization), to
identify where within a cell or within
a tissue a particular protein is
located.
17. A technique that employs
antibodies is western blotting. In
this technique, a mixture of proteins
is separated by SDS-PAGE. The
proteins in the gel are transferred

and bound directly to a membrane, which is then treated with
an antibody specific for one of the proteins. The membrane is
washed to release unbound antibody

and then the antibody-protein complex
is visualized. This can be by a
fluorescent technique or more
commonly by an enzyme linked to the
antibody as above. In any case, the
protein of interest is identified in this
way.
18. MALDI-TOF is a mass
spectometric analysis instrument that
facilitates determination of polypeptide
masses with great accuracy. It
employs a laser, which, when
activated, causes a polypeptide
sample to volatilize in the evacuated
chamber of the instrument.
Volatilization causes the molecules to
both ionize and to break at peptide
bonds. Masses are determined by the
length of time it takes for the ions to
travel through the chamber to the
detector. The time it takes them to
make the transit (Time of Flight= TOF)
is proportional to their mass. Smaller
fragments move faster than larger
fragments. Using MALDI-TOF, it is
possible to determine the sequence of
amino acids in a polypeptide based on
the analysis of the masses of the
various fragments that arise from ionization.
255
19. X-ray crystallography is a technique whereby a crystal of a

pure protein (or DNA or RNA) is subjected to beams of x-rays.
The movement of x-rays through the crystal is aected by their
interaction with electron clouds, causing the x-rays to be bent
or deflected. By studying the angles of deflection, one can,
using sophisticated
computer analysis,
determine the electron
density of the material
the x-rays passed
through. The electron
density map gives 3D
coordinates of every
atom in the crystal to
within a few
Angstroms.
256
6. If one plots the percentage of oxygen sites bound versus

partial pressure for myoglobin, a hyperbolic curve is generated,
1. Myoglobin and hemoglobin are related
consistent with a molecule with a single
proteins involved in (respectively) storing
binding site and a high anity for
A
worried
young
scientist
reckons
and carrying oxygen in the body.
oxygen. The P50, which is the partial
He must do something when aging beckons
pressure of oxygen necessary to fill 50%
2. Myoglobin is a single subunit protein with
So hell make himself younger
of the myoglobins with oxygen, is very
By developing hunger
high anity for oxygen. It holds oxygen
low for myoglobin, consistent with high
So then he can go back four seconds
tighter than hemoglobin and serves in a
anity. Because myoglobin has high
battery-like capacity in tissues to release
anity for oxygen, it doesn't release much oxygen until it is in
oxygen when tissue oxygen concentration is very low.
an environment with very low oxygen pressure. For this reason,
Myoglobin can take oxygen from hemoglobin.
it would be a poor oxygen transport protein.
3. Hemoglobin is a four-subunit protein complex (two alpha
7. Hemoglobin is much better designed to meet an organism's
subunits and two beta subunits) that serves to carry oxygen
physiological needs for transporting oxygen than myoglobin.
from the lungs to the tissues where it is needed. Hemoglobin is
This is due to its four-subunit organization (one heme per
genetically related to myoglobin and is evolutionarily derived
subunit and one oxygen carried per subunit) which behaves in
from it.
a cooperative fashion in binding oxygen.
4. Myoglobin and hemoglobin both have porphyrin rings (like in
8. Binding of oxygen by the iron atom causes it to be pulled 'up'
chlorophyll) to hold ferrous (Fe++) iron. The specific porphyrin
slightly. This, in turn, causes the histidine attached to it to
in these proteins is Protoporphyrin IX. Ferrous iron is the form
change position slightly, which causes all
of iron involved in carrying oxygen. Ferric iron
the other amino acids in the subunit to
I do not think it is a good sign
(Fe+++), which is the oxidized form of iron will
Im so busy that I must decline
change slightly. The changes in shape
not carry oxygen. Heme is a term used to
The demand of my boss
(dierent 'states') result in the protein
describe the protoporphyrin IX complexed with
To get the watch that she lost
gaining anity for oxygen as more oxygen
iron.
Because I just cant find the time
is bound. The phenomenon is referred to as
5. The iron in hemoglobin and myoglobin is held
cooperativity.
in place by five molecules - four nitrogens of the protoporphyrin
9. Hemoglobin can exist in a "tight" state, called 'T', which
IX ring and a histidine (called the proximal histidine). Oxygen is
exhibits low oxygen binding anity. Hemoglobin in the T state
carried between the iron and an additional histidine (called the
will tend to release oxygen.
distal histidine) not involved in holding the iron.
Key Points - Hemoglobin
257
10. A second state of hemoglobin is the "relaxed" or R state,

which exhibits increased oxygen binding anity. Binding of
oxygen by hemoglobin helps it to "flip" from the T to the R
state and release of oxygen by hemoglobin helps it to flip from
R to T.
11. A molecule called 2,3-bisphosphoglycerate (2,3 BPG) is
produced by actively respiring tissues. It can bind in the gap in
the center of the hemoglobin molecule and in doing so,
stabilize the T state and favor the release of oxygen. Thus,
tissues that are actively respiring get more oxygen. 2,3 BPG is
noteworthy because the blood of smokers has a higher
concentration of the molecule than the blood of non-smokers.
12. Fetal hemoglobin diers from adult hemoglobin in having two
gamma subunits in place of the two beta subunits that adults
have. This changes the hemoglobin molecule such at 2,3BPG
can't bind, so fetal hemoglobin exists more in the R state and
has a higher anity for oxygen than adult hemoglobin. Thus,
the fetus can take oxygen from the mother's hemoglobin, due
to this property. The fetus won't release oxygen as readily as
the adult, but it doesn't have a high oxygen need, due to not
exercising heavily.
13. The Bohr eect describes physiological and molecular
responses to changes in pH with respect to oxygen and carbon
dioxide in the body. The oxygen eects arise from changes in
the tertiary structure of hemoglobin arising from binding of
protons to histidines in the molecule when under low pH.
14. Rapidly metabolizing tissues (such as muscle) generate lower

pHs, due to release of carbon dioxide and its conversion to
carbonic acid by carbonic anhydrase. Carbonic acid readily
258
loses a proton,
becoming
bicarbonate.
15. Thus, rapidly
metabolizing tissues
generate protons,
which get absorbed
by hemoglobin, which
releases oxygen to
feed the tissues.
16. CO2 can also be
taken up by
hemoglobin at amine
residues, causing
protons to be
released. Note that
CO2 binds
hemoglobin at a site
other than what
oxygen binds. CO,

however, can
compete with oxygen for binding to the heme.
17. In the lungs, a reversal of this process occurs. Remember that
the oxygen concentration in the lungs is high, so oxygen forces
o the carbon dioxide and the carried protons from the
hemoglobin. Addition of a proton to bicarbonate re-creates
carbonic acid, which undergoes the reversal of the earlier
carbonic anhydrase reaction, causing CO2 to be released to the
lungs as a gas.
18. Sickle cell anemia is a

genetic malady that results in
a hemoglobin that polymerizes
under low oxygen conditions,
causing blood cells to form
sickle shapes. These can get
stuck in capillaries (painful)
and the cells can be removed
by the body as possibly
damaged, resulting in anemia.
19. Sickle cell anemia appears
to provide protection against
malaria.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
259
Key Points - Enzymes

1. Enzymes are proteins that catalyze
reactions.
2. Enzymes are capable of speeding
reactions quadrillions of times faster than
the same reactions would occur in the
absence of enzymes.
biological catalysts) act by lowering G+. Catalysts DO NOT

CHANGE G. All they do is lower the energy required to
activate the reaction. While enzymes speed
At the hamburger stand there was one
reactions immensely, they therefore DO
Young lady who did an end run
NOT CHANGE THE OVERALL REACTION
Around regulations
CONCENTRATION AT EQUILIBRIUM. They
And caused a sensation
simply allow the reaction to get to
By putting her hair in a bun
equilibrium faster.
3. Non-proteinaceous molecules that bind to enzymes and help

the enzymes to catalyze reactions are called coenzymes.
4. The Gibbs free energy is the energy available to do useful work
in reactions. The change in the Gibbs free energy for a reaction
is important because it determines whether a reaction is
favored (G <0), unfavored (G >0), or at equilibrium (G = 0).
5. Thus, when the Gibbs free energy change is negative, the
reaction in question goes forward as written, but when the
Gibbs free energy change is positive, the reaction goes in
reverse.
8. G is aected by the concentration of reactants and products

of a reaction by the following equation
G = G' + RTln{[Products]/[Reactants]}
(Note, this assumes a single reactant and a single product)
Thus, as product concentrations increase, the G will become
more positive.
9. If one performs an experiment in which a fixed amount of

enzyme is added to 20 dierent tubes, each containing a
dierent amount of substrate (molecule that the enzyme
catalyzes the reaction on) and then lets the reaction in each
6. A related term to G is G', which is the
tube go for a fixed amount of time, one will
Standard Gibbs Free Energy change. This
In choosing a theme for the rowers
create varying amounts of product when the
refers to the Gibbs Free Energy change for
The adman gave what they asked for
tubes are analyzed. The greatest amount of
A clever clich
a reaction under standard conditions. Since
product will be found in the tube which had
He
inverted
to
say
most reactions occur at non-standard
the greatest amount of substrate. If one
No
ifs,
ands
or
buts,
just
oars
conditions, G is much more useful than
measures the concentrations of each product
G'. In fact, the sign of G' does NOT tell
and divides by the time the reaction occurred,
the direction of a reaction, except under standard conditions.
one obtains a velocity for each reaction. A plot of the velocity
versus the substrate concentration (V versus S) from the
7. Chemical reactions require activation energy (we'll call it G+
experiment looks like the binding curve of myoglobin for
here) in order to get started. Catalysts (both enzymes and non260
The cement man complained with great ardor

To his boss, I dont think its smarter.
Everything that I do
To satisfy you
Just keeps getting harder and harder.
oxygen -
hyperbolic.
10. Velocity of an enzymatic reaction is measured as the
concentration of product formed per time. Maximum velocity
(Vmax) occurs in a reaction when the enzyme is saturated with
substrate. Vmax depends on the amount of enzyme used to
measure it.
11. In contrast to Vmax, Kcat is a constant for an enzyme. It is also
known as the turnover number and corresponds to the number
of molecules of product made per molecule of enzyme per time
(usually in seconds). 1000/second means 1000 molecules of
product per molecule of enzyme per second.
12. We define Km as the substrate concentration that gives Vmax/2.
Whereas the Vmax varies, depending on the amount of enzyme
that one uses, the Km is a constant for a given enzyme for its
substrate.
13. The higher the Km of an enzyme, the LOWER its anity for its
substrate. This is because a high Km means that it takes a LOT
of substrate before the enzyme gets to Vmax/2. Km is frequently
referred to as the anity of the enzyme for a substrate, though
that is not 100% correct. Nevertheless, we say that a high Km is
consistent with a low anity of enzyme for substrate and
conversely, that a low Km is consistent with a high anity of
enzyme for substrate.
14. If one lets a reaction go for a long time, it will reach

equilibrium. At equilibrium, the relative concentration of
products and reactants do not change. Initial velocities of
reactions are therefore measured so as to avoid allow the
product to accumulate and favor the reverse reaction.
15. Lineweaver-Burk plots are alternative plots of V vs S data
obtained by taking the inverse of each and plotting it, thus
making a 1/V vs 1/[S] plot (also called a double reciprocal plot).
16. On a Lineweaver-Burk plot, the Y intercept is 1/Vmax and the X
intercept is -1/Km.
17. The catalytic actions of enzymes appear to be related to their
ability to be at least slightly flexible. Originally, Fischer
proposed a model of catalysis called the Lock and Key model.
It described enzymes as inflexible and the substrate as like a
key fitting into a lock. While substrates do, in fact, fit into
enzymes somewhat like a key, the enzyme is NOT rigid.
18. Koshland's model of enzyme action, called the Induced Fit
model says that not only does the enzyme change the
substrate (via catalysis), but the substrate also changes the
enzyme shape upon binding. This transient change of enzyme
shape is important for catalysis because it may bring together
molecular groups (such as a phosphate and a sugar) that may
not be close together in the enzyme prior to the change in
enzyme shape.
Remember that
Perhaps it was heavenly love
Or some coincidences thereof
enzymes also work by
That gave Matt a B
orienting substrates
In trigonomtry
together in the proper
He thinks twas a sine from above
way to maximize their
261
likelihood of bouncing together in a way that leads to making a

bond.
19. Chemical changes brought about by catalysis facilitate a last
change in enzyme shape to allow for the release of the
products. When this happens, the enzyme returns to its original
shape and remains unchanged by acting to catalyze the
reaction.
When the pant leg got torn to extremes
Its owner was left without jeans
A seamstress could make
A new leg in a shake
Or perhaps just at least sew its seams
20. "Perfect"
enzymes are
enzymes that
have evolved to
the point where
any additional
mutation will reduce their ability to catalyze reactions. They are
not common. Perfect enzymes have a very high ratio of Kcat/Km
and are such that the only thing that inhibits their ability to
function more eciently is the rate of diusion of substrate in
water.
21. The active site of an enzyme is the place in the enzyme where
a reaction is catalyzed. A substrate is a molecule bound by an
enzyme and acted upon it.
22. Substrate binding to enzymes is relevant to catalysis that we
will consider. The first is the category of Sequential
Displacement. It has two subsets.
a. Random binding - the order of binding multiple substrates
is not rigidly set.
b. Ordered binding - Simple ordered binding - one substrate

binds first followed by another followed by release of
product.
23. Note that the models of substrate binding above are both
non-covalent.
24. Another non-covalent enzyme mechanism involving multiple
substrates is called Double Displacement (or Ping-Pong
kinetics). In this method, the enzyme only binds one substrate
at a time, but switches back and forth between dierent states.
In the exam given in class, transaminase flips back an forth
between carrying an oxygen or carrying an amine group. This
also aects what substrate it binds to.
25. Allosterism is a phenomenon in which a small molecule
interacts with a protein and aects the proteins activity. Such
an enzyme is an
allosteric enzyme.
The tert alcohol was a fool
There is similarity
Turned to ketone in making a fuel
between the kinetics of
The work is complete
allosterically acting
The bond gave up heat
As it lost all its family joules
enzymes and the
cooperativity of binding
of oxygen by hemoglobin.
26. Enzymatic reactions can be inhibited by reversible and
irreversible processes. Reversible processes involve binding of
an inhibitor and its subsequent release. Irreversible processes
generally involve covalent attachment of a molecule to an
enzyme followed by its inactivation.
262
27. Competitive Inhibition This type of reversible

inhibition occurs when
the inhibitor competes
with the substrate for the
binding site on an
enzyme. The greater the
concentration of inhibitor,
the greater the inhibition.
However, competitive
inhibition can be
overcome by increasing
amounts of substrate.
The apparent Vmax of
competitive inhibition
does not vary from the
Vmax of the same reaction
when uninhibited. The
apparent Km, however,
does vary, because it
requires more substrate

to get the same velocity
as the uninhibited reaction.
28. Non-competitive inhibition - This type of reversible inhibition
occurs when an inhibitor binds to an enzyme at a site unrelated
to the substrate binding site. In this case, the inhibitor's binding
to the enzyme is unrelated to the binding of the substrate and
the inhibitor does not have to have a structure like that of the
substrate. Thus, the inhibitor and substrate don't compete with
each other. The inhibitor can inhibit the enzyme (during its
binding) without
interference from the
substrate. Therefore,
increasing substrate
concentrations cannot
eliminate the eect of the
inhibitor. In this case, the
Vmax is lowered, but the
Km is unchanged.
29. Students should be
able to depict or
understand graphically (V
vs S, Lineweaver-Burk)
what occurs in competitive
and noncompetitive
inhibition.
30. Chemicals, such as
DIPF and iodoacetate,
covalently (and irreversibly)
bind to the side chains of
specific amino acids (serine
and cysteine, respectively) and if these side chains are
essential for the catalytic action of the enzyme, the enzyme
may not catalyze reactions after being treated with these
chemicals.
At the circus it is common sense
31. Penicillin is a
If a fire breaks out your defense
substance that
Should be to be scopin'
resembles the
The places most open
substrate of an
Because heat will be most in tents
263
enzyme in
bacteria that
helps make the
bacterial cell
wall. When it
binds to the
enzyme, it inactivates the enzyme by covalently bonding to the
active site, thus destroying the enzyme (and killing the
bacterium containing it). An inhibitor of this type is known as a
suicide inhibitor.
Politicians may find it profound

But we hope that they will come around
To this simple advice
For keeping things nice
Throwing dirt means youre just losing ground
32. There are six classes of reactions catalyzed by enzymes. They

include Oxidation-Reduction Reactions (electrons gained/lost),
Ligation Reactions (two molecules put together), Isomerization
Reactions (intramolecular rearrangements), Group Transfer
Reactions (movement of a part of one molecule to another
molecule), Hydrolytic Reactions (breakdown reactions using
water), and Lyases (breakdown reactions involving a double
bond, but not using water to break the molecule down).
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
264
3.When the release of the colored substrate by the

enzyme is studied, it appears to occur in two dierent
rates. First there is a VERY rapid release of the colored
substrate. After that initial burst of activity, the remaining
yellow color is released slowly.
4.The reason appears to be that the reaction catalyzed
occurs in two steps. The first step cleaves the bond to
produce the yellow product, which is rapidly released.
The other product of this reaction is the remainder of the
substrate that is covalently linked to the enzyme. In order
for the enzyme to bind another substrate molecule and
release more yellow color, it must first release the
covalently bound molecule. This step occurs slowly and
explains why subsequent yellow molecules are released
slowly - after the initial one is released, the enzyme must
remove the covalently bound molecule, bind a new
substrate, and cut the substrate and the continue the
process repeatedly.
Key Points - Catalytic Strategies

1. Proteases catalyze the hydrolysis of peptide bonds in
polypeptides. They are usually fairly specific for certain amino
acids and cut at or near those amino acids.
2. Chymotrypsin is a protease whose activity has been closely
studied. Conveniently, the activity of chymotrypsin can be
studied using an artificial substrate which, when cleaved by the
enzyme, releases a yellow product.
5.Chymotrypsin is an example of a protease that

employs reactive serine in its active site. Such an enzyme
is called a serine protease. Treatment of chymotrypsin
with DIPF, which covalently links to serines, inactivates the
enzyme.
6. Serine proteases form covalent intermediates with their
polypeptide substrates. The first step involves nucleophilic
attack of an alkoxide ion on the polypeptide substrate to form
an acyl-enzyme intermediate. Formation of this intermediate
results in cleavage of the peptide bond and release of the first
polypeptide fragment. The acyl-enzyme intermediate is
265
resolved by addition of water to release the other

portion of the original polypeptide along with
regeneration of the original enzyme active site. This
last step occurs relatively slowly.
7. In the active site of chymotrypsin (and other serine
proteases) is a so-called catalytic triad of amino acids
that includes a serine hydrogen bonded to a histidine.
The histidine is, in turn, hydrogen bonded to an
aspartic acid residue in the active site. Each of the
hydrogen bonds of the catalytic triad is important in
the catalytic mechanism. The nucleophilic alkoxide ion
on serine is made possible ultimately by interactions in
the catalytic triad and these hydrogen bonds.
8. The catalytic triad is not unique to chymotrypsin. The
serine protease known as subtilisin also has the same
catalytic triad and employs a similar mechanism.
9. Besides the catalytic triad, the enzyme has two other
important sites to consider. The first is the oxyanion
hole that stabilizes a tetrahedral intermediate that
arises during the catalysis. The second, called as S1

pocket, is where the substrate binds. Both the oxyanion hole
and the S1 pocket are adjacent to the active site (catalytic
triad).
I confess with a bit of hostility
I succumbed due to some gullibility
My info got took
By a mean online crook
Possessing a great lie ability
10. Many other

proteases and
other enzymes
share similarities
with the
mechanism used
by serine proteases. Other proteases include cysteine

proteases (use cysteine and histidine in the active site), aspartyl
proteases (use aspartic acids and water in the active site)
metalloproteases (use a metal ion - usually zinc - and water in
the active site).
11. Cysteine proteases use an ion of the sulfhydryl group of
cysteine to act as a nucleophile to attached the carbonyl
peptide bond and facilitate breakage of the peptide bond.
266
12. Aspartyl
The magician would bid all adieu
proteases
Disappearing from audience view
employ two
At the end of the session
There was only one question
aspartic acid
Was that just a stage he went through?
side chains
to hold water
in place and use an ion of one of them to act as a nucleophile
to attack the peptide bond.
13. Metalloproteases use a metal ion to hold water in place so it
can be ionized to act as a nucleophile to attack the peptide
bond.
14. Carbonic anhydrase is an enzyme that catalyzes the joining of
carbon dioxide and water to form carbonic acid.
15. A zinc ion (held in place by three histidines in the active site of
carbonic anhydrase) plays an important role in the catalysis of
the enzyme by binding a water molecule. A subsequent loss of
a proton by water is necessary for catalysis. Notably, the
enzyme has maximal activity at a high pH (where protons are
easily removed) and a lower activity in an acidic pH (6.0).
16. The limiting step in the action of carbonic anhydrase is the
abstraction of the proton from water. Buers and/or bases help
facilitate this and thus speed the reaction.
17. Restriction enzymes are bacterial enzymes that can cleave
DNA by breaking phosphodiester bonds between adjacent
nucleotides in the molecule.
"pocket" for a water molecule and magnesium ion to be

positioned properly so that the water can be activated to be a
nucleophile to attack the phosphodiester bond between
nucleotides to cleave it.
19. Restriction enzymes are paired with a methylase in bacterial
cells. The methylase puts a methyl group on the same
sequence the restriction enzyme would otherwise cut. When
cellular DNA is protected in this way, the restriction enzyme
cannot cut the cellular DNA, but it can cut invading viral DNA if
it gets to it before the methylase does.
20. Restriction enzymes bind to DNA and "slide" along the double
helix. When they reach the sequence they cut at (recognized by
hydrogen bonds), the DNA is bent and a magnesium ion is
allowed into the complex to facilitate the activation of water to
nucleophilically attack the phosphodiester bond, cleaving it.
21. Myosin is a protein that undergoes a significant shape change
upon hydrolysis of ATP. This action allows it to move along
actin filaments and facilitates muscular contraction.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
18. Restriction enzymes act at specific nucleotide sequences.

When these bind in the enzyme, it changes shape and this
shape change causes a bend in the DNA. The bend provides a
267
Key Points - Allostery and Regulation

1. Aspartate transcarbamoylase (ATCas) is an enzyme that
catalyzes the first step in pyrimidine biosynthesis (aspartate +
carbamoyl phosphate <=> N-carbamoylaspartate). This
enzyme is allosterically regulated in both a
positive and negative fashion and also
responds to the binding of the substrate
(aspartate) to it.
2. CTP, the end product of pyrimidine
the enzyme has increased activity and a higher anity for

substrate. ATP stabilizes the R state of the enzyme.
5. In the absence of ATP and CTP, the enzyme can freely flip
between the T and R states, but the T state tends to
predominate.
I had asked to try out something new

On the burger I ordered from you
Perhaps you got flustered
When adding the mustard
Because it seems like Dijon Vu
6.If one removes all of the ATP and CTP from

the enzyme and starts adding increasing
amounts of the enzyme's normal substrate
(aspartate) and then measures the reaction
biosynthesis, inhibits the enzyme, whereas ATP (a purine and
rate (velocity), one discovers a sort of sigmoidal plot, which
an indicator of high energy) activates the enzyme. This
indicates that the enzyme is actually changing as more
phenomenon - where the end product of a metabolic pathway
aspartate is added to it. The actual change is a flip from the T
inhibits the first enzyme in the pathway - is known as feedback
state to the R state stabilized by the aspartate.
inhibition. Feedback inhibition is mediated allosterically - when

a small molecule binds to a protein and aects the protein's
activity.
3. ATCase has 12 subunits - 6 catalytic and 6 regulatory. The
smaller regulatory subunits bind CTP, but not the catalytic
subunits.
4. Binding of CTP to the regulatory subunits of ATCase causes
7. Students should be familiar with the eects of ATP, CTP and

increasing aspartate on ATCase and where they bind on the
enzyme to exert their eects.
8. A manmade artificial substrate, known as PALA, binds to
ATCase and inhibits the enzyme, but interestingly locks it in the
R state. Though the enzyme can't catalyze a reaction when
bound to PALA, it is clear that its form changes, again
the enzyme to stabilize (lock) in the T state (tight, less reactive
indicating that
state). In the T state, the quaternary structure of the enzyme
enzymes are changed
the enzyme exhibits reduced anity for substrate. In the
by the binding of
opposite state, the R state (relaxed state, more reactive state),
substrate and that T

and R states exist.
When the food color was misapplied

To the meat loaf the baker supplied
The patrons ingested
But barely protested
Even though they were dyeing inside
268
9. Protein kinase A is an enzyme involved in covalent

modification of enzymes. Like all kinases, it catalyzes the
addition of a phosphate to a molecule. Since it is a protein
kinase, the molecules it attaches phosphate to are proteins.
10. Phosphates get
become active. When active, they covalently modify proteins

by adding phosphates.
12. Zymogens are enzymes that are synthesized in an inactive
form whose activation requires covalent modification, usually
proteolytic cleavage. Examples
attached to hydroxyl
include digestive enzymes, such
side chains in proteins.
as trypsin, chymotrypsin,
Protein kinase A
elastase, and carboxypeptidase
attaches phosphate to
whose enzymatic activity might
serine or threonine.
be harmful to the tissue where
Other protein kinases
they are being made. Trypsin is
can attach phosphate
the primary activator of an entire
to tyrosine side chains.
class of proteolytic enzymes.

Improper activation of trypsin in
11. Protein kinase A is
or close to the pancreas can
controlled by allosteric
lead to pancreatitis, which arises
means. It is composed
when the proteases attack
of two regulatory
proteins in the pancreas.
subunits and two

catalytic subunits. The
13. Activation of
overall complex is
chymotrypsinogen to
called R2C2. When the
catalytic subunits are
chymotrypsin, for example,

requires trypsin. Trypsin makes
bound to the regulatory subunits, they cannot catalyze
an initial cleavage between amino acids 15 and 16. A disulfide
reactions. However, when the molecule cAMP binds to the
bond keeps the two pieces from coming completely apart,
regulatory subunits, the catalytic subunits are released and
however. This creates an intermediately active form of

chymotrypsin called pi-chymotrypsin. This form cleaves itself
269
to remove two dipeptides and this results in full chymotrypsin
fibrinogen (zymogen) to fibrin (mesh former as a result of
activity. The three polypeptide pieces are held together by
polymerization).
disulfide bonds. Note that three very minor changes in the

chymotrypsinogen zymogen have converted a completely
inactive enzyme to a fully active one.
14. Alpha-one-antitrypsin is an inhibitor of many proteases,
17. Prothrombin must bind calcium in order to be held near the

site of the wound to be activated. Binding of calcium by
prothrombin allows it to anchor itself to the phospholipid
membranes derived from blood platelets after injury. At this
including trypsin and elastase. Normally alpha-one-antitrypsin
site, prothrombin can be readily converted to thrombin because
binds to elastase and prevents it from damaging tissue. People
at the site of the injury are other enzyme that can activate
deficient in the factor have proteins in their alveolar walls of the
prothrombin to thrombin. To enable prothrombin to strongly
lung (and other connective tissue proteins) destroyed,
bind calcium, glutamate residues in it must be carboxylated
ultimately resulting in emphysema. Smoke from cigarettes will
(addition of a carboxyl group). This reaction is catalyzed by an
react with HEALTHY alpha-one-antitrypsin, causing a
enzyme that uses Vitamin K as a cofactor. Compounds like
methionine residue to be oxidized, which, in turn, prevents
coumarin or warfarin that block vitamin K sites on the enzyme
alpha-one-antitrypsin from binding to elastase, allowing
act as "blood thinners", reducing the likelihood of blood
elastase to damage alveolar tissue and eventually cause
clotting by inhibiting the carboxylation of prothrombin.
emphysema.
15. Blood clotting is a process that is also tightly controlled by
18. Removal of blood clots involves an enzyme called plasmin,

which is synthesized as a zymogen called plasminogen.
zymogens. The process is a cascade of catalytic events in
Plasminogen is converted to plasmin by tissue-type
which one enzyme activates another, which in turn activates
plasminogen activator (t-PA). t-PA can be extremely eective in
hundreds/thousands, which in turn activate millions. Cascades
initiating the cascade to dissolve the unwanted blood clot.
are great for amplifying a signal.

16. Two pathways activate the blood clotting process, the
Jump to Chapter
intrinsic pathway and the extrinsic pathway. Ultimately these

cascades lead to the conversion of prothrombin (zymogen) to
thrombin (active enzyme). Thrombin, in turn, converts
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
270
Key Points - Carbohydrate

Nomenclature, Structure and Function
1. Simple carbohydrates are monosaccharides, also called
sugars. These include glucose, galactose, and mannose.
asymmetric) and the groups can be arranged in two dierent

ways. These arrangements are called stereoisomers.
6. The letters 'D' and 'L' are an older nomenclature system used
to designate whether a particular stereoisomer rotated
polarized light rightwards or
2. The sux '-ose' is used
leftwards, respectively. We
to designate
use the convention today
saccharides. The
that the 'D' isomer
number-related prefixes
corresponds to the
'tri', 'tetr', 'pent', 'hex',
stereoisomer in which the
'hept', and 'oct' are used
lowest asymmetric carbon
to designate saccharides
from the top (closest to the
with 3,4,5,6,7, and 8
bottom) is written on the
carbons, respectively.
right side of the molecule.
3. Monosaccharides with
7. Most biological sugars
an aldehyde group are
are in the D configuration.
called aldoses. Those

with a ketone group are
8. Stereoisomers, such as
called ketoses.
D-glyceraldehyde and Lglyceraldehyde, are mirror
4. Glyceraldehyde and
images of each other.
dihydroxyacetone are the

simplest saccharides we
call carbohydrates.
5. Carbon can have as many as four dierent molecular groups
attached to it. If this happens, the carbon is chiral (or
9. When sugars dier in

stereoisomeric
configuration, they are called diastereomers. When
stereoisomers are mirror images of each other, they are called
enantiomers. When they dier in configuration of only one
271
carbon, they are called epimers. When they dier only in the
reversibly form. Thus, a sugar in the beta configuration can, in
configuration of the anomeric carbon, they are called anomers.
solution, 'flip' to the alpha form by going to the linear form and
then reverting back to the ring structure in the alpha
10. Cyclization of
configuration. If the
monosaccharides leads
hydroxyl of the anomeric
typically to five member
carbon is altered (by
or six member rings.
methylation, for example),
These are called
the linear structure cannot
furanoses (five member
form and 'flipping' cannot
rings) and pyranoses (six
occur.
member rings).
Cyclization arises from
13. Altering the hydroxyl
formation of hemiacetals
group on the anomeric
in aldoses and
carbon results in creation of
hemiketals in ketoses.
a glycoside. Glycosides are

commonly created during
11. Haworth structures
formation of disaccharides
refer to the ring forms of
and longer carbohydrates.
sugars. The straight

chain forms are referred
14. A given sugar such as
to as a Fischer
beta-D-glucose can have
projections.
dierent conformations that
12. Cyclization creates a

new asymmetric carbon. This carbon is called the anomeric
carbon and it can exist in the alpha (down position) or beta (up
position) configurations. If the hydroxyl group on the anomeric
have dierent shapes.

These are referred to as 'boat' and 'chair' forms. Chair forms
are generally favored over boat forms due to less steric
hindrance.
carbon is unaltered, the ring and linear forms of the sugar can
272
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Thatthe business was bad
OnlineOrigami has folded
15. Linking together of
more than one sugar residue). Homopolymers include glycogen
more than one sugar
(glucose in alpha 1-4 linkages plus extensive alpha 1-6
residues creates
branches), cellulose (glucose in beta 1-4 linkages), amylose
higher order
(glucose in alpha 1-4 linkages), amylopectin (glucose in alpha
saccharides. These
1-4 linkages plus some alpha 1-6 branches), and chitin (N-
include disaccharides (two sugars), trisaccharides (three
acetyl-D-glucosamine in beta 1-4 linkages).
sugars), oligosaccharides (several sugars), and

polysaccharides (many sugars).
16. Most of the linkages in higher order saccharides
involve glycosidic bonds.
17. Disaccharides include sucrose (glucose +
fructose), lactose (glucose + galactose), and
maltose (two glucoses).
18. Sucrose is a non-reducing sugar (has no free
anomeric hydroxyl), whereas lactose is a reducing
sugar (has a single free anomeric hydroxyl).
19. Oligosaccharides are components of
glycoproteins.
20. The most common polysaccharides include
glycogen (energy storage in animals), cellulose
(structural integrity in plants), starch (energy storage
in plants), chitin (exoskeleton of insects).
21. Polysaccharides can be homopolymers (contain
only one sugar residue) or heteropolymers (contain
273
22. Glycogen is an animal energy storage

polysaccharide, amylopectin and amylose
combine to form starch, which is a plant
energy storage polysaccharide, cellulose
is a plant structural polysaccharide, and
On the latest test I was confused

By the math trigonometers use
It went rather fast
I hope that I passed
I dont have a secant to lose
27. Oligosaccharides on proteins (glycoproteins)

and lipids (glycolipids) have functions in cellular
identity and can be recognized and bound by
immunoglobulins.
28. Oligosaccharides on the surface of cells help
chitin is a component of insect exoskeletons.
give them their identity. For example, A,B, and O blood group
23. The enzyme cellulase is required to digest the beta 1-4 bonds
of cellulose. Most animals do not contain cellulase. Ruminants
antigens give rise to the various blood types and these arise
from carbohydrates on their cell surfaces.
and ungulates contain the bacterium that makes that

enzyme.
24. Glycosaminoglycans are polysaccharides that contain
either N-acetylgalactosamine or N-acetylglucosamine
as one of their monomeric units. They are polyanionic
and have interesting chemical properties, as a result.
Examples include chondroitin sulfates and keratan
sulfates of connective tissue, dermatan sulfates,
heparin, hyaluronic acid, and others.
25. Proteoglycans are complexes of proteins and
glycosaminoglycans that form feathery structures.
26. Glycoproteins consist of a protein linked to an
oligosaccharide, usually via an 'N' or an 'O' linkage. N
linkages occur through asparagine of the protein. O
linkages occur across serine or threonine of the
protein.
274
29. Transplanted organs suer rejection when the new organ

has a dierent oligosaccharide pattern than the organ the
recipient originally had. This encourages the immune system
to attack it as foreign.
30. All N-linked glycoproteins have the same core of five
carbohydrate residues. N-linked glycoproteins have
glycosylation (addition of carbohydrate residues) occurring
in the endoplamic reticulum and Golgi complex of the cell.
O-linked glycoproteins have glycosylation occuring only in
the Golgi complex.
31. Movement of modified proteins from the endoplasmic
reticulum to the Golgi complex allows for additional
carbohydrate modifications to occur, followed by targeting
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Gave it all with an old college try
The results in the end
Did their future portend
Since they both ended up in a tie
to 1) the cell
membrane, 2)
release from the
cell, or 3) the
the sialic acid o with a neuraminidase enzyme (also on the flu
lysosome.
virus's surface). Anti-flu drugs like tamiflu act by inhibiting the
32. Oligosaccharides destined to be linked to proteins to make
action of the neuraminidase.
glycoproteins are "built" on dolichol phosphate on the outer

portion of the endoplasmic reticulum and then this "flips" to the
inside for attachment.
Jump to Chapter
33. Specific carbohydrate residues on the surface glycoproteins

of blood cells are binding targets for hemagluttanin proteins on
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
the surface of flu viruses. To enter a cell, the virus must cleave
275
Key Points - Signaling

1. Signaling is essential for cells in multicellular organisms to
activated PKA begins phosphorylating a set of enzymes,

turning them on or o (depending on the enzyme).
3. Phosphorylation of proteins known as transcription factors can
communicate with each other.
activate or inactivate them. When activated they will turn on
2. A common signaling system
transcription of specific
is that of the beta-
genes in the DNA.
adrenergic receptor, which

works as follows. A ligand,
4.Thus, signaling can
such as epinephrine (also
have rapid eects
called adrenalin) is released
(controlling enzyme
into the bloodstream in
activities) or slower
response to a stimulus. This
eects (controlling gene
molecule is called the first
expression by controlling
messenger. When it arrives
transcription).
at the target cell, it binds to
5. Receptors of the first
the receptor, causing the
messenger have
receptor to change shape
similarity in structure,
slightly. The result of this
consisting of a
shape change is that on the
polypeptide chain that
inside of the cell, the
spans the cell
receptor acts on a G protein

(see below) to activate it.
The activated G protein, in
membrane 7 times. Such
turn, activates the enzyme

adenylate cyclase, which, in turn, begins to catalyze synthesis
of cAMP. cAMP is a so-called second messenger, which acts
by binding to Protein Kinase A (PKA) to activate it. The
proteins are called 7TM

proteins.
6. G proteins get their name from the fact that they bind guanine
nucleotides (GDP and GTP). G protein complexes have three
subunits - alpha, beta, gamma. Cells have 'families' of G
276
protein complexes arising from the fact that there are multiple,
involves phosphorylation of the receptor (by a receptor kinase).
slightly dierent versions of the individual subunits in the
The phosphorylated receptor is then bound by the protein
genome and these can be paired in many ways. The alpha
called beta-arrestin that binds the receptor and inhibits the
subunit binds to GDP or GTP. When the alpha subunit is bound
activation of G proteins.
to GDP, it also binds the beta and gamma subunits. The G

protein complex is thus inactivated. When the beta-adrenergic
receptor binds its ligand, the receptor stimulates the 'loading'
of GTP onto the alpha subunit, displacing GDP in the process.
Upon binding GTP, the alpha subunit dissociates from the beta
and gamma units. The alpha subunit is then free to bind other
target proteins and is thus 'active' when bound to GTP.
7. G proteins have an enzymatic activity that slowly breaks down
9. cAMP in cells acts

on a kinase known
as Protein Kinase
A (PKA). PKA is
composed of four
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The optometrists zen mental state
Theres no big surprise
It includes both the eyes
Get up early, work hard, and dilate
subunits - two
identical catalytic subunits (C) and two identical regulatory
subunits (R). When the complex is present as C2R2, the PKA is
GTP to GDP within the alpha subunit. This activity is very
inactive. Binding of cAMP to the R subunits causes them to
important because it ensures that the G protein will not be left
dissociate from the C subunits. The freed C subunits are
in the 'on' state permanently. When the alpha subunit is bound
therefore active.
with GTP, it can bind to adenylate cyclase (an enzyme

embedded in the cell membrane) and activate it. When the
alpha subunit has GDP,
In the sea it can get quite complex
Tracking down the particlars of sex
If an 8-legged guy
Likes his mom on the sly
Should they call him an Octopus Rex?
it is bound with the

beta/gamma subunits
and CANNOT bind to
adenylate cyclase.
8.Cells have two ways
of turning o the beta adrenergic receptor. The first involves

simple dissociation of the epinephrine ligand from the receptor.
This leaves the receptor in the 'o' state. The second method
10. Signaling through the adrenergic receptor has the eect of

increasing blood glucose. Insulin is a hormone that counters
the eect of epinephrine. It should be noted that increasing
concentration of cAMP in cells results in an increase of blood
glucose. Phosphodiesterase breaks down cAMP. Inhibitors of
phosphodiesterase, such as caeine, have the eect of
increasing blood glucose.
11. Other receptors involved in signal transduction (signaling) act
in dierent ways. For example, some receptors stimulate the
activity of the enzyme Phospholipase C. This enzyme acts on a
277
membranous molecule called phosphatidyl inositol (or PIP2).
precipitating DNA. Calcium is essential for muscular
Cleavage of PIP2 by phospholipase C results in production of
contraction.
TWO second messengers. One of these, diacylglycerol (DAG)

remains near or in the lipid bilayer where it stimulates another
kinase, Protein Kinase C.
13. EF Hands are important structural domains of calcium binding

proteins. Calmodulin is one such protein.
Protein Kinase C acts to
14. Calmodulin binds
phosphorylate numerous
calcium, helping to keep its
proteins/enzymes to
concentration relatively low.
activate/inactivate them,
Upon binding calcium,
depending upon the
calmodulin changes shape
enzyme. The other
and this change in shape
second messenger
allows it to bind to other
produced by
proteins that it wouldn't
phospholipase C
otherwise be able to bind
cleavage is inositol 1,4,5
to. One class of these are
triphosphate (also called
the CaM kinases that are
IP3). IP3 is soluble in the
stimulated to phosphorylate
cytoplasm and it acts
proteins when calmodulin
there to stimulate the
binds.
release of calcium from
15. Another signaling
intracellular storage
mechanism is mediated
areas holding calcium.
12. Calcium may be
directly through receptors

and doesn't involve G
thought of as a kind of 'third' messenger in the process of
proteins. A good example is the insulin receptor. This receptor
signaling. Cells normally must keep the concentration of the ion
is normally present in the membrane of a target cell in a dimeric
low so as to prevent it from binding to proteins and
form and consists of two extracellular alpha subunits and two

278
intracellular beta subunits. The beta subunits contain the active
19. PIP3 in the membrane is the binding target for PDK1 (PIP3-
site of the enzyme, a tyrosine kinase. In the absence of insulin
dependent protein kinase). PDK1 is thus activated in this way
binding, the subunits are non-phosphorylated and the kinase is
and it phosphorylates another kinase known as Akt. Akt is NOT
inactive.
membrane bound and upon phosphorylation is activated. It can
16. Binding of insulin by the insulin receptor moves the units of

the dimer closer
together. This, in
turn, causes the
two tyrosine
kinase sites to
In their schools little fishies await

The subject they all think is great
They dont line in a path
To sign up for math
Instead all of them take debate
then move through the cell, phosphorylating other proteins and

activating pathways. One of the pathways it activates results in
the movement of the protein known as GLUT4 to the cell
surface. GLUT4 is a glucose transport protein that transports
glucose into cells.
20. Another signaling system is that going through the epidermal
phosphorylate tyrosine residues on the opposite one, activating
growth factor (EGF) receptor. Epidermal growth factor is a small
it. The two kinases then phosphorylate tyrosines at other
polypeptide whose action stimulates cells to divide and grow.
places on each beta subunit.

17. The phosphorylated tyrosines on the insulin receptor are
binding targets for proteins known as insulin receptor
substrates (IRS-1). The protein contains a common domain,
called SH2 that recognizes and binds phosphorylated tyrosines.
Many proteins have SH2 domains.
18. When IRS-1 binds to the insulin receptor, it gets
phosphorylated on tyrosines, as well, and these
phosphorylated tyrosines are bound by the SH2 domain of an
enzyme known as phosphatidylinositide-3-kinase. When
phosphatidylinositide-3-kinase binds to the IRS protein, it is
stimulated to phosphorylate PIP2 in the membranes, making
PIP3.
The EGF receptor that

My dog has received a bad mark
On his driving test out near the park
He drove with both paws
And obeyed all the laws
But just couldnt parallel bark
it binds to exists in cell

membranes as a
MONOMER. Binding of
EGF to the monomeric
receptor causes the
receptor to dimerize.
21. The EGF receptor, like the insulin receptor, has an intracellular
domain that is a tyrosine kinase. Dimerization of the receptor
causes the carboxy-terminal portion of the receptor to become
phosphorylated at tyrosines.
22. Genes in which mutations can happen that lead to
uncontrollable growth are known as oncogenes. The
279
unmutated forms of these genes perform important signaling or

control functions. These unmutated genes are known as protooncogenes.
23. Ras is a proto-oncogene. Mutations of ras that interfere with
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
its ability to cleave GTP to GDPcan lead to uncontrolled cellular

growth.
24. One type of leukemia (CML) arises from DNA rearrangement
that joins together two gene-coding sequences known as BCR
and ABL. ABL is a tyrosine kinase that plays an important role
in controlling when cells divide. When BCR is joined to ABL
(making BCR-ABL), the fusion that arises is made in greater
quantities than ABL itself and it still contains the tyrosine kinase
activity. Thus, the overexpressed fusion stimulates cells to
divide uncontrollably.
25. BCR-ABL tumors are successfully treated with a tyrosine
kinase inhibitor known as Gleevec.
The sailor is singing with ease

On his ship in a rather strong breeze
For his kind of a song
He can draw a big throng
Every time that he hits the high seas
280
Key Points - Metabolic Control

1. Metabolism is literally the chemistry of life. Schematic
diagrams of metabolism are the equivalent of highway
maps for a city. Central pathways, like glycolysis, are the
equivalent of the main streets.
2. To understand chemical reactions in cells, we must
understand the thermodynamics (energy) of the reactions.
3. The free energy of a process is the energy available to do
useful work. The free energy of a process is called the
Gibbs Free Energy.
4. Most commonly, we are concerned with the change in free
energy for a system. For example, for an enzymatically
catalyzed reaction, we are interested in the change in free
energy between the reactants and the products. The
change in free energy (G) indicates the favorability of a

process.
a. If the G for a process is negative, the process is favored.
b. If the G for a process is positive, the process is not
favored (in fact, the reverse of the process is favored).
Mister Bach was a wonderful bloke
Making music that oft would evoke
A satisfied smile
Dramatic in style
Thank goodness he went for baroque
c. If the G for
a process is
Example: For A <=> B,

if G is negative, A->B is favored.
if G is positive, B-> A is favored.
if G is 0, B-> A and A-> B are equally favored. The system
is at equilibrium.
5. The chemical potential of a component A is equal to the
zero, the
chemical potential at the standard state plus RT ln[A]. For a
process is at
reaction of multiple components
equilibrium.
281
aA + bB <=> cC + dD
available energy. The higher the oxidation state of a molecule,

the less energy that can be obtained from it. Thus, glucose,
6. G is the G measured under standard conditions (all

products and reactants at 1M). Here,
G = G
7. For biological systems, we define a G
(note the prime at the end) to define
standard conditions for aqueous
which has a higher oxidation state than fatty
A violation of Sir Isaac was found

acids, provides less energy to cells than fatty
By young Megan on her way to the ground
acids.
Shes not in Smithereens
Because on trampolines
12. ATP is made from ADP by phosphorylation.
What goes down must go up then go down
There are three types of cellular phosphorylation
in nature. Substrate level phosphorylation
solutions at pH 7.0.
8. ATP is a source of energy in cells because the G of
the hydrolysis reaction is very negative (releases much
free energy). Hydrolysis of ATP directly to yield energy
(like burning of wood to heat a house) is not the
mechanism used by cells to drive reactions. Instead,
hydrolysis of ATP is coupled to energetically
unfavorable reactions to make them proceed.
9. Cells must have ATP to accomplish work (muscular
action), transmit information (nerve signals), signal
each other (cellular signaling), and synthesize
important biochemicals.
10. Oxidation is used to provide the energy necessary to
make ATP. ATP energy is used to provide reduction
necessary to biosynthesize compounds like fats and
fatty acids.
11. The oxidation state of a molecule is related to its
282
occurs when high energy
15.For every oxidation (loss
phosphate molecules
of electrons) by one
(like creatine phosphate)
molecule, there is a
transfer their phosphate
reduction (gain of electrons)
directly to ADP to form
by another one. In
ATP. Oxidative
biological systems, electron
phosphorylation occurs
carriers, such as NAD+/
as a result of actions in
NADH and FAD/FADH2 are
the mitochondria,
electron carriers. When a
ultimately caused by
biological molecule is
oxidation.
oxidized, electrons are
given (in pairs) to either
(occurs only in
NAD+ or FAD to form NADH
photosynthetic
or FADH2. NADH and
organisms) uses light
FADH2 can also donate
energy to make ATP.
electrons to biological
13. Creatine phosphate is a

backup ATP source for
molecules, thus reducing
muscle cells. This arises from the following reaction

the biological molecules.

16. An example oxidation/reduction reaction that might occur in
cells is
(G = + 12 kJ/mol)
Alcohol + NAD+ <=> Aldehyde/Ketone + NADH + H+
14. Normally when at rest, this reaction moves to the RIGHT, due
17. Catabolism generally involves oxidation and/or breakdown of
to high ATP concentrations. Upon exercising, however, ATP
large molecules (proteins, fats, carbohydrates) into smaller
concentrations drop, and the reaction moves to the left,
ones (many of these converge at acetyl-CoA). Catabolism
restoring ATP levels, at least temporarily.
releases energy that is used to make ATP. Anabolism generally

283
involves reduction and/or synthesis of large molecules from
the synthesis of large molecules from small ones is not
small ones. Anabolism requires an energy source - often from
energetically favorable, but when energy from ATP is provided,
ATP.
they become energetically favorable.
18. ATP energy in anabolism is used to drive energetically
19. Besides ATP energy (or GTP or UTP or CTP, as appropriate),
unfavorable reactions by coupling the hydrolysis of ATP (an
other mechanisms available to cells to "drive" reactions
energetically favorable process) with the reaction that is
forward are to alter the concentrations of reactants and
energetically unfavored (such as the addition of phosphate to
products. We define the phenomenon of "pushing" a reaction
glucose, as noted in class). By doing this coupling, an
as increasing the amount of reactants. This has the eect of
unfavorable reaction becomes energetically favorable. Thus,
reducing the G value and making a reaction more favorable

in the forward direction. We also define the phenomenon of
"pulling" a reaction as decreasing the amount of product. This
too has the eect of reducing the value of G and favoring a
reaction to go forward.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
284
Key Points - Glycolysis &

Gluconeogenesis
1. Glycolysis, the breakdown of glucose, is a catabolic pathway
involving oxidation and yields ATP and NADH energy.
Gluconeogenesis, the
3. In reaction #1 of glycolysis, hexokinase catalyzes transfer of

phosphate to glucose from ATP, forming glucose-6-phosphate,
G6P. Thus, this step uses ATP, which provides the energy
necessary for the reaction to proceed. It is an example of an
energy-coupled reaction and the G is strongly negative, thanks
to the ATP hydrolysis.
synthesis of glucose,
is an anabolic
4. Hexokinase changes
pathway that involves
shape as it binds to glucose.
reduction and uses
This property is consistent
ATP and NADH. There
with that of an induced fit of
are 10 reactions in
an enzyme in the process of
glycolysis. Students
catalysis.
should know
5. Reaction #2 of glycolysis
structures of fructose
is catalyzed by
and glucose
phosphoglucoisomerase. In
compounds, all
it, G6P is converted to F6P.
enzyme names, all
The G for the reaction is
molecule names, and
close to zero.
reactions where the
6. Reaction #3 is the primary
G is strongly
positive, or strongly
negative.
regulatory reaction of
2. Note that glycolysis has two phases - an energy investment

phase requiring input of ATP energy and an energy realization
phase where ATP is made.
glycolysis. It is catalyzed by
phosphofructokinase (PFK).
Note that this reaction also requires ATP. PFK is the most
important regulatory enzyme for glycolysis. The molecule made
in the process, F1,6BP, is a high energy molecule and the
285
At the bookstore I asked timidly

For some help to get phobia-free
The clerk pointed to
A brand new review
But I fear that it may not help me
energy in the molecule
catalyzed by glyceraldehyde-3-phosphate dehydrogenase
is needed in the next
(G3PDH). The energy of the oxidation is used to put phosphate
reaction. The reaction
onto the acid produced by the oxidation. The products are
is another example of
NADH and 1,3 BPG. The latter has high energy (higher than
an energy-coupled
ATP).
reaction and the G is strongly negative, thanks to the ATP

hydrolysis.
11. Reaction #7 is catalyzed by phosphoglycerate kinase and it

includes a substrate level phosphorylation (transfer of a
7. Reaction #4 is catalyzed by aldolase. It has a strongly positive
phosphate from a molecule directly to ADP) to make ATP.
G. In the cell, however, the reaction is pulled by

reactions ahead of it (which remove products) and
pushed by reactions behind it (which increase amounts
of reactants), making the G favorable (negative). The
products of this reaction are G3P and DHAP.
8. The energy barrier of reaction 4 is overcome by
'pushing' (increasing concentration of reactatnts) and
'pulling' (decreasing concentration of products) of the
reaction.
9. Reaction #5 is catalyzed by the 'perfect' enzyme
known as triose phosphate isomerase. The reason the
enzyme operates so fast is to prevent accumulation of
a toxic intermediate. The product of the reaction is
G3P. The G is close to zero, so this reaction is
readily reversible. Everything after this step has two
molecules of each.
10. Reaction 6 is the only oxidation in glycolysis. It is
286
12. Reaction #8 is catalyzed by phosphoglycerate mutase and it
15. The phenomenon of redox balancing is important for
simply involves rearrangement of the 3PG into 2PG. The G
glycolysis. Redox balancing relates to the relative amount of
is close to zero and the direction of the reaction is driven by
NAD+ and NADH in the cell. Remember that reaction 6 is very
cellular concentrations. Note that the mutase creates as an
sensitive to the ratio of NAD+/NADH.
intermediate 2,3BPG, which is the molecule hemoglobin bound

to and caused it to release oxygen.
16. Pyruvate has three separate fates, depending on conditions

and the cell type. When
13. Reaction #9 is catalyzed by
oxygen is present, there is
enolase and involves removal of
plenty of NAD+, so aerobic
water from 2PG to form PEP, which
cells convert pyruvate to
is a highly energetic compound.
acetyl-CoA for oxidation in

the citric acid cycle. When
14. Reaction #10 is the "Big Bang" of
oxygen is absent, NAD+
glycolysis. It is catalyzed by the
levels can go down, so to
enzyme pyruvate kinase and in the
prevent that from happening,
reaction, a substrate level
pyruvate is converted to
phosphorylation yields ATP. Note
either lactate (animals) or
that the G is very strongly
ethanol (bacteria/yeast).
negative, helping to pull all the
Either of these last two
reactions preceding it to a large
conversions requires NADH
extent. The enzyme is allosterically

inactivated by ATP and allosterically
activated by F1,6BP. The latter

activation is an example of "feed forward" activation. Pyruvate
kinase is also inactivated by phosphorylation, as will be seen in
glycogen metabolism.
and produces NAD+. NAD+

made in this way can be used
to keep reaction 6 of glycolysis going.
17. Anaerobic conversion of NADH to NAD+ provides much less
ATP energy to cells than when oxygen is present. Anaerobic
metabolism of glucose generates only 2 ATPs per glucose,
287
The Asian chef worked with aplomb

For a name for his restaurants dot com
He got most creative
Sticking close to the native
And dubbed it the Viet Nom Nom
whereas aerobic
in the human eye lens causes it to absorb water and this may
metabolism of
be a factor in formation of cataracts.
glucose generates
38 ATPs per
glucose.
18. Other sugars than glucose can be metabolized by glycolysis,

if they are converted to intermediates of glycolysis.
19. Entry of fructose to the glycolysis cycle may be problematic in
some cases. Fructose can be converted to F6P by hexokinase.
Fructose can also be converted to fructose-1-phosphate (F1P)
by fructokinase. Conversion of F1P to glyceraldehyde and
DHAP allows fructose to be metabolized by glycolysis without
being controlled by PFK. Ingestion of a lot of fructose (via high
fructose corn syrup in many foods) may be a factor in obesity.
20. Other sugars than glucose can be metabolized by glycolysis,
if they are converted to intermediates of glycolysis.
21. Galactose can enter glycolysis by being converted to
galactose-1phosphate followed by conversion (ultimately) to
glucose-1-phosphate and subsequently to glucose-6phosphate, which is a glycolysis intermediate.
22. Deficiency of galactose conversion enzymes results in
23. Deficiency of the enzyme lactase leads to lactose intolerance

24. Regulation of glycolysis is controlled by three enzymes hexokinase, PFK, and pyruvate kinase. Hexokinase's regulation
is a bit complicated and is controlled partly by availability of
substrate.
25. PFK is very unusual in being negatively regulated by a
molecule (ATP) that is also a substrate. This is possible
because the enzyme has an allosteric binding site for ATP in
addition to the substrate binding site and the Km for the
allosteric site is higher than the substrate binding site.
26. Pyruvate kinase is regulated both allosterically and by
covalent modification (phosphorylation/dephosphorylation).
Phosphorylation of the enzyme by a protein kinase turns the
enzyme activity down, whereas F1,6BP acts as an allosteric
activator. This activation is known as feedforward activation.
27. Feedforward activation is rare in metabolism. It is a term used
to describe a metabolic product (such as F1,6BP above) that
ACTIVATES an
enzyme that
accumulation of galactose (from breakdown of lactose). Excess
catalyzes a
galactose is converted to galactitol (a sugar alcohol). Galactitol
reaction further
ahead of it in a
If his hair was not tightly held on

Captain Kirks message might be withdrawn
And replaced with a thought
That would not be forgot
Baldly going where no man has gone
288
metabolic pathway. In glycolysis, feedforward activation acts to
HIF-1, as well.
start the pyruvate kinase reaction and PULLs the reactions
Another way
forward to get over the energy "hump" of the aldolase reaction.
cancer cells battle
28. Hypoxia refers to the condition where cells are short of

oxygen. Since oxygen is necessary for maximum energy
production from glucose, they must respond to this condition.
One way they respond is by making a transcription factor
known as Hypoxia Induction Factor 1 (HIF-1). HIF-1 activates
transcription of genes involved in glucose transport and
glycolysis. Cancer cells are frequently hypoxic and induce
hypoxia is to
stimulate the
The cable guy felt quite impelled

In the wedding his wife and he held
To get everything right
He would work overnight
Making sure the reception excelled
growth of blood vessels to them by making another factor

known as angiogenin. Blocking HIF-1 and angiogenin are anticancer therapies.
29. Glycerol is a breakdown product of fat metabolism. Glycerol
can be metabolized in glycolysis by conversion to glycerol-3phosphate and then to DHAP, a glycolytic intermediate.
30. Gluconeogenesis accomplishes the reverse of glycolysis synthesis of glucose from pyruvate using four dierent
enzymes to replace three energetically unfavorable reactions in
glycolysis.
31. Gluconeogenesis does not occur in all tissues of the body.
The primary gluconeogenic organs of the body are the liver and
part of the kidney.
32. The enzymes unique to gluconeogenesis are Pyruvate
Carboxylase and PEP carboxykinase (PEPCK) instead of
Pyruvate Kinase of glycolysis, Fructose 1,6 Bisphosphatase
(F1,6BPase) instead of Phosphofructokinase (PFK) from
glycolysis, and Glucose-6-phosphatase (G6Pase) instead of
Hexokinase from glycolysis.
289
33. F1,6BPase and G6Pase act by similar

mechanisms, clipping a phosphate from their
substrates and thus avoiding synthesis of ATP,
which is what would be required if the glycolysis
reactions were simply reversed.
In the forest you should be aware

Flexibilitys really quite rare
Youd be surprised catching
Any wildlife out stretching
Except for a few yoga bears
make glucose in gluconeogenesis. The

glucose is then dumped into the bloodstream,
where it travels back to the muscles that need
it.
37. Anabolic and catabolic pathways
34. One reaction of gluconeogenesis occurs in the mitochondrion.
occurring at the same time and place create a futile cycle.
It is catalyzed by pyruvate carboxylase and yields the four
Futile cycles generate heat, but that is the only product they
carbon intermediate, oxaloacetate. The carboxyl group is
make. Consequently, cells usually set up controls that turn one
added in forming oxaloacetate thanks to the coenzyme biotin,
o when the other is turned on. If the same molecule has
which carries carbon dioxide for attachment. The remaining
opposite eects on catabolic and anabolic pathways, the
reactions all occur in the cytoplasm, except for the G6Pase
molecule is a reciprocal regulator of the pathways. Reciprocal
reaction, which occurs in the lumen of the endoplasmic
regulation of catabolic and anabolic pathways is a very ecient
reticulum.
means of control.
35. Anabolic and catabolic pathways occurring at the same time
38. The enzymes of glycolysis that are regulated have
and place create a futile cycle. Futile cycles generate heat, but
corresponding gluconeogenesis enzymes that are also
that is the only product they make. Consequently, cells usually
regulated. PFK and F1,6BPase exhibit the most complicated
set up controls that turn one o when the other is turned on. If
regulation. Both are controlled by several mechanisms. The
the same molecule has opposite eects on catabolic and
most important one is the allosteric reciprocal regulation by
anabolic pathways, the molecule is a reciprocal regulator of the
fructose-2,6-bisphosphate (F2,6BP). F2,6BP activates PFK and
pathways. Reciprocal regulation of catabolic and anabolic
inhibits F1,6BPase.
pathways is a very ecient means of control.

36. The Cori Cycle is a cycle of the body where
the lactate of working muscles is dumped
into the bloodstream. It travels to the liver
where it is converted to pyruvate and used to
39. F2,6BP is made and degraded by two

On the most recent summer vacation
Only one place deserved condemnation
Its dry summer breezes
Gave us too many sneezes
When we got to our first dusty nation
dierent portions of the same protein

(We'll use PFK2 to refer to the kinase
portion and FBPase-2 to refer to the
phosphatase portion). The portion of the
290
40. Phosphorylation of the enzyme by protein kinase A is

favored by 7TM signaling whereas dephosphorylation by
phosphoprotein phosphatase is activated by signaling by
insulin.
41. Thus phosphorylation of the PFK2 favors the
breakdown of F2,6BP and the activation of
gluconeogenesis and deactivation of glycolysis.
Dephosphorylation of PFK2 favors the synthesis of
F2,6BP and the activation of glycolysis and the
deactivation of gluconeogenesis. This is the heart of
reciprocal regulation of these pathways.
42. Pyruvate kinase, pyruvate carboxylase, and PEPCK
are all regulated, as well. Pyruvate kinase is activated by
feedforward activation by F1,6BP and is inhibited by ATP
and alanine (a product easily made from pyruvate).
Pyruvate kinase is also controlled by covalent
PFK2 catalyzing the synthesis of F2,6BP from F6P is PFK2. The

portion of the protein catalyzing the breakdown of F2,6BP to
F6P is FBPase-2. The two activities are regulated by
phosphorylation of the PFK2/FBPase-2 protein by protein
modification as described in the previous highlights.

Phosphorylation of the enzyme makes it less active,
whereas dephosphorylation make it more active.
Jump to Chapter
kinase A. When phosphorylated, the PFK2 part of the enzyme

is inactive and the FBPase-2 is active. When the phosphate is
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
removed from the protein by phosphoprotein phosphatase, the

PFK2 becomes active and the FBPase-2 becomes inactive.
291
Key Points - Glycogen Metabolism

1. The structure of glycogen consists of units of glucose linked in
the alpha1-4 configuration with branches linked in the alpha1-6
configuration.
5. Glycogen phosphorylase action on glycogen yields glucose-1phosphate. Glycogen phosphorylase exists in two forms phosphorylase a and phosphorylase b. Phosphorylase a diers
from phosphorylase b only in that phosphorylase a contains
two phosphates and
2. Glycogen diers from
phosphorylase b contains
amylopectin in the
none. Phosphate is added
amount of branching
to glycogen phosphorylase
(glycogen has much
by the enzyme
more).
phosphorylase kinase.
3. Glycogen is a storage
6.Glucose-6-phosphate
form of energy that can
(G6P) has many dierent
yield ATP very quickly,
fates and sources. First,
because glucose-1-
breakdown of glycogen
phosphate can be
produces G1P, which is
released very quickly.
readily converted to G6P.

G6P can then go three
4. Students should know
dierent directions. In
the function/activities of
muscle and brain (and most
the enzymes in glycogen
other tissues), G6P enters
breakdown - glycogen
glycolysis. In liver only, G6P
phosphorylase,
phosphoglucomutase,
and debranching
enzyme.
enters gluconeogenesis
and is converted to glucose

for export to the bloodstream. In other tissues, G6P enters the
pentose phosphate pathway and is oxidized to produce
NADPH.
292
7. Breakdown of glycogen by glycogen phosphorylase involves

phosphorolysis (use of a phosphate to cleave molecules)
instead of hydrolysis. The advantage of this is
that the energy of the alpha1-4 bond is used
to add phosphate to glucose (forming G1P)
instead of using a triphosphate to do so. This
saves energy for cells.
by a 'side-step' reaction that creates uridine diphosphate

glucose (UDP-Glucose or UDPG) from G1P.
The ocular surgeon can please

All the ladies with his surgeries
Though ecient at working
He is quite slow at flirting
That uncrossing eyes dodding tease
8. Glycogen phosphorylase catalyses phosphorolysis of glycogen

to within 4 residues of a branch point and then stops. Further
metabolism of glycogen requires action of debranching
enzyme. Debranching enzyme removes three of the remaining
11. Synthesis of UDPG requires UTP and G1P

and produces UDPG and pyrophosphate (PPi).
UDPG is made by action of the enzyme UDPG
pyrophosphorylase.
(In the notes below, we refer to Glycogen
Phosphorylase a as GPa and Glycogen Phosphorylase b as GPb.

We also refer to Glycogen Synthase a as GSa and Glycogen
Synthase b as GSb.
four glucoses at a branch point and transfers them to another
12. Glycogen phosphorylase is present in two covalently dierent
chain in an alpha1-4 configuration. The remaining glucose in
forms, GPa and GPb. They dier in phosphorylation. GPa is
the alpha1-6 configuration at the branch point is cleaved in a
phosphorylated and GPb is not.
hydrolysis reaction to yield free glucose. It is the only free

glucose released in glycogen metabolism.
13. GPb is converted into GPa by phosphorylation at two sites.

Covalent modifications are DIFFERENT from allosteric controls,
9. Phosphoglucomutase interconverts G1P and G6P via a G1,6BP

intermediate. The reaction is readily reversible (G near zero)
which interconvert the R and T states of BOTH GPa and GPb.

14. The dierent glycogen phosphorylase forms have dierent
and the direction of the reaction depends

on the concentration of substrates.
10. Synthesis of glycogen is not the simple
reversal of the steps in glycogen
breakdown. There is an energy barrier
The camping group at the Great Lakes

Think their planners are really big flakes
The cause of dissent?
They cant pitch a tent
Because of too many missed stakes
that must be overcome - synthesis of the alpha1,4 bond

between adjacent glucoses in glycogen. This is accomplished
allosteric regulation, as well. GPb is

converted to the R state by AMP and
converted to the T state by ATP and G6P.
The latter two are more commonly
abundant in the cell than AMP, so we think
of GPb as less active. GPa is converted to
the T state by glucose (rarely present much). When glucose is

293
absent, GPa is automatically in the R state. Thus, we think of
glycogen to yield G1P

17. Glycogen synthase (catalyzes formation of glycogen) exists in
GPa as the more active form.

15. Phosphorylation of GPb to make GPa is catalyzed by an
enzyme known as phosphorylase kinase. Full activation of
phosphorylase kinase requires both calcium ions and
phosphorylation by protein kinase A
16. Binding of epinephrine to the cell surface stimulates the
following events in muscle relating to glycogen breakdown
two dierent covalent forms - GSa (has no phosphate and is

the most active) and GSb (has phosphate and is the least
active).
18. Binding of epinephrine or glucagon to a cell surface receptor
ultimately results in the phosphorylation of GPb and GSa to
make GPa and GSb, respectively. The products of these
phosphorylations - GPa
A. Epinephrine binds
receptor
and GSb have opposite
B. Receptor activates a
G protein to bind GTP
ACTIVE and GSb is
activities. GPa is MORE

LESS ACTIVE. Thus,
C. Alpha subunit of G
protein activates
adenylate cyclase
reciprocal regulation of
glycogen metabolism is
D. Adenylate cyclase
catalyzes formation of
cAMP
mediated through
phosphorylation and
dephosphorylation. The
E. cAMP activates
protein kinase A
conversion of R and T
states occurs
F. Protein kinase A
phosphorylates
phosphorylase kinase,
activating it.
allosterically.
19. Reversal of the
process (removal of the
G. Phosphorylase kinase
phosphorylates GPb,
converting it to GPa
H. GPa breaks down
phosphates of all of the

phosphorylated
enzymes) occurs as the

294
I went o to my grocer to beg

Lest I drop cooking standards a peg
The source of the fuss?
The high price of nuts
They are costing an almond a leg
result of catalysis by
phosphoprotein
phosphatase (PP-1).
PP-1 is stimulated to be
active by insulin. Thus,
increasing glucose
concentration in the bloodstream causes insulin to be released,
phosphorylating additional glycogen phosphorylase enzymes.

22. Insulin is capable (via binding to a cell surface receptor) of
reversing the action of the phosphorylation system. It does this
by stimulating the activity of Protein Phosphatase I (PP1). This,
in turn, causes ALL OF THE EARLIER PHOSPHORYLATIONS
TO BE REVERSED.
which causes cells to take up glucose and also (inside the cell)
to cause the glucose to be made into glycogen.
20. Reversing phosphorylations causes glycogen
breakdown to cease and glycogen synthesis to begin.
Remember that insulin is released in response to an
increase in blood sugar and it stimulates cells to take
up glucose. Thus, when the cells take up glucose, the
glycogen synthesis system is stimulated to put it into
glycogen and this occurs because insulin stimulates
the activity of Protein Phosphatase (PP1), which is
capable of removing phosphates from all the proteins
described above.
21. So, when insulin binds the cell surface receptor,
glycogen synthesis is stimulated (glycogen synthase is
converted from the 'b' form to the 'a' form) and
glycogen breakdown is inhibited (glycogen
phosphorylase is converted from the 'a' form to the 'b'
form). In addition, PP1 removes the phosphate from
glycogen phosphorylase kinase, which stops it from
295
23. By contrast, the

protein kinase A
phosphorylation
system
simultaneously
The troubled young sprinter did vow

Shed make an adjustment somehow
Though the hurdles she feared
All were nonetheless cleared
Its good that shes over them now
activates glycogen
breakdown (by making GPa) and inhibits glycogen synthesis
(by making GSb), it also INACTIVATES the enzyme that
removes phosphates (PP1).
GPa, forming GPb. Freed from GPa, can then PP-1

dephosphorylate GSb, forming GSa. As a result, glycogen
synthesis is NOT activated until glycogen breakdown is first
stopped.
27. Thus, addition of glucose to purified GPa and GSb causes
conversion of GPa to GPb and GSb to GSa. Addition of
glucose causes GPa to flip into the T state, which causes it to
release PP-1-GL to begin dephosphorylation of the two
enzymes.
24. PP-1 binds to a protein called GM (in muscle) or GL (in liver).

When bound to GL, PP-1 is held close to the glycogen
phosphorylase, which is useful because this allows easy
Jump to Chapter
access to dephosphorylate it and turn it o.

25. Activation of PKA by epinephrine or glucagon causes GM to
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
be phosphorylated, which, in turn, causes PP-1 to be released

in a less active form. PKA also phosphorylates the PP-1
inhibitor, which then binds PP-1 and inactivates it. Thus, the
phosphorylation system shuts down the dephosphorylation
system and vice versa, depending on which hormone has
bound to the cell surface receptor.
26. GPa normally binds PP-1-GL tightly and acts as a glucose
sensor in liver cells. PP-1 is inactive when bound to GPa if GPa
is in the R state. Increasing glucose concentration causes GPa
to flip into the T state. When GPa is in the T state, PP-1-GL is
released from GPa, becomes active, and dephosphorylates
296
Key Points - Citric Acid Cycle

1. Both oxidative decarboxylation (in higher cells) and nonoxidative decarboxylation (in yeast) use an enzymatic activity
called the pyruvate dehydrogenase complex to convert
2. It also uses five coenzymes, Thiamine Pyrophosphate (TPP),

Lipoamide, NAD+, FAD, and Coenzyme A (also called CoASH
or CoA).
3. The mechanism of the reaction catalyzed by the complex is
pyruvate from glycolysis
very similar to that
into acetyl-Coa for the
catalyzed by the alpha-
citric acid cycle. This
keto-glutarate
enzyme complex is in the
dehydrogenase complex of
mitochondrion and
the citric acid cycle. Both
requires that pyruvate
involve oxidation of alpha-
from the cytoplasm be
keto acids.
transported to the
4. In aerobic higher
mitochondrion. This
organisms, the reaction
complex includes the
mechanism involves
following:
binding of pyruvate by an
ionized TPP,
a. Pyruvate
decarboxylase
decarboxylation, transfer to
(some books call it
the lipoamide molecules,
"Pyruvate
linkage of the acetyl group
Dehydrogenase
to CoASH to form acetyl-
Component" (E1)
CoA, transfer of the
b. Dihyrolipoamide
transacetylase (E2)
electrons from the oxidation

to FAD (forming FADH2) and
transfer of electrons from FADH2 to NAD+ to form NADH.
c. Dihyrolipoamide dehydrogenase (E3)

297
The Shakespearean critic was shrill

With the language he used to fulfill
His role as complainer
And major disdainer
That man was a fier at Will
5.In yeast fermentation,

the reaction that occurs
stops at the
decarboxylation step
with resolution to form
acetealdehyde without loss/gain of electrons (no oxidation/

reduction). Thus, enzyme activities E2 and E3 above are not
needed in yeast fermentation. Acetaldehye in yeast
fermentation is converted to ethanol by reduction, using
electrons from NADH. Note that when oxygen is present,
9. The citric acid cycle consists of two main parts - release of CO2
(first part) and conversion to oxaloacetate (second part).
10. In the "first" reaction of the citric acid cycle, citrate synthase
catalyzes the joining of the acetyl group from acetyl-CoA to
oxaloacetate to make citrate. This reaction is VERY
energetically favorable, due to breaking of the thioester bond in
acetyl-CoA. The energetically favorable reaction helps to "pull"
the relatively unfavorable reaction preceding it.
11. Aconitase catalyzes the rearrangement of citrate to isocitrate.
fermentation in yeast does not occur and activities E2 and E3
Aconitase is inhibited by fluorocitrate. Fluoroacetate is a poison
catalyze reactions just like animal cells, producing acetyl-CoA.
that can be used by citrate synthase to make fluorocitrate.
6. Mitochondria are the "power plants" of the cell and are the
places where much oxidation occurs.
7. The citric acid cycle occurs in the mitochondrial matrix (inner
portion of mitochondrion) and is found in almost every cell. In
the cycle, two carbons are added from acetyl-CoA and two
carbons are released as carbon dioxide.
8. Biological oxidation of intermediates in the citric acid cycle
involve NAD+ (reduced to NADH) and FAD (reduced to FADH2).
In the citric acid cycle, three NADH and one FADH2 are
produced, along with one high energy phosphate (GTP in
animals, ATP in plants and bacteria) per acetyl-CoA that enters
the cycle (Remember that one molecule of glucose yields two
acetyl-CoAs for the cycle).
12. The first decarboxylation of the citric acid cycle is catalyzed

by isocitrate dehydrogenase and the reaction is strongly
favored to the right. The products of this reaction are NADH
and alpha ketoglutarate.
13. Alpha ketoglutarate is an important intermediate for its
involvement in anaplerotic reactions related to transamination.
The mechanism of the enzyme acting on alpha ketoglutarate
(alpha ketoglutarate dehydrogenase complex) is virtually
identical to the
mechanism of
action of the
pyruvate
dehydrogenase
My love for the stu is insane

Its addicting almost like cocaine
I guess Im a softie
For freshly shipped toee
That arrives in a big chew chew train
complex and
298
involves all of the same coenzymes. The products of this

reaction are succinyl-CoA and NADH.
14. The only substrate level phosphorylation in the citric acid
16. Addition of water to fumarate (catalyzed by fumarase) yields

L-malate.
17. Oxidation of L-malate by malate dehydrogenase yields NADH
cycle is catalyzed by
and oxaloacetate. This
succinyl-CoA
reaction is a rare
synthetase. The
oxidation reaction that
products of this reaction
is energetically
in the citric acid cycle
unfavorable. Conversion
are GTP and succinate.
of malate to
Note that the enzyme is
oxaloacetate is the only
named for the reverse
energy "bump" to be
reaction.
gotten over in the citric

acid cycle and that is
15. Succinate
readily accomplished
dehydrogenase
thanks to the 'pulling' of
contains a covalently-
the citrate synthase
linked FAD electron
reaction, which keeps
carrier. The G of zero
oxaloacetate
allows the reaction to
concentrations low.
be readily reversed to
produce succinate,
when needed. The
18. The citric acid cycle

can be regulated
products of this reaction in the forward direction of the citric
allosterically in several places, but the most important
acid cycle are FADH2 and fumarate (trans double bond). This
regulation of the cycle is probably the amount of NAD+ and
reaction is similar to the first oxidation reaction for a fatty acid.
FAD that is available. NAD+ (and FAD) is essential for the cycle
to operate and it is essential for the pyruvate dehydrogenase
complex reaction to occur. This relates to metabolic control, as
299
will be more obvious in the sections on electron transport and
Remember that NAD+ is required for three reactions of the citric
oxidative phosphorylation.
acid cycle. If any one of these reactions is stopped, the cycle
19. When all of the NADHs and FADH2s of the citric acid cycle are
converted to ATP, the cycle yields 30- 38 ATPs per molecule of
grinds to a halt. While glycolysis can use fermentation to get

around conditions lacking oxygen, the citric acid cycle cannot.
glucose (depending on how
21. The pyruvate
you count them), compared
dehydrogenase complex
to 2 for glycolysis (under
is regulated by substrate
anaerobic conditions). The
level regulation (NAD+/
citric acid cycle is thus an
NADH) and allosteric
incredibly ecient producer
regulation (ATP
of energy for the cell.
inactivates, AMP and

ADP activate). The
20. Many factors combine to
complex is also
regulate metabolism
regulated by covalent
through the citric acid cycle.
modification -
All of these ultimately come
phosphorylation/
down to energy needs.
dephosphorylation. The
Most are manifested
relevant enzymes are a
through the availability or
kinase, which
lack of NAD+. When NAD+ is
phosphorylates pyruvate
lacking (high NADH levels),
dehydrogenase
the cycle will be inhibited.

When NAD+ levels are high
(low NADH levels), the cycle

is favored. Oxygen is a limiting reagent needed to keep the
citric acid cycle turning. This is because oxygen is required
ultimately for the conversion of NADH back to NAD+.
(inactivating it) and a

phosphatase, which
dephosphorylates pyruvate dehydrogenase, activating it.
22. Pyruvate dehydrogenase is sensitive to arsenite and mercury,
due to the fact that these compounds will react with the sulfur
300
The puzzle was clear on the shore

When the crew teams most talent rower
Got caught in a bind
To make up his mind
When he just couldnt pick either oar
atoms in lipoamide of
25. The glyoxylate cycle is a pathway related to the citric acid
E3 of the pyruvate
cycle that occurs in plants and bacteria. It requires two
dehydrogenase
enzymes not found in animals (in addition to the normal
complex. Treatment
enzymes of the citric acid cycle). The enzymes unique to the
of lipoamide with BAL
glyoxylate cycle are isocitrate lyase (catalyzes cleavage of
extracts arsenite from

the lipoamide, due to the fact that BAL also contains sulfurs to
isocitrate to glyoxylate and succinate) and malate synthase

(catalyzes linkage of acetyl-CoA to glyoxylate to form malate).
which the arsenite binds.

23. Anaplerotic reactions are involved in "filling up" the
intermediates of metabolism, which are needed for
multiple purposes. For example, oxaloacetate may be
used up in making aspartic acid and must be
replenished. One mechanism for this may be
conversion of glutamic acid into alpha ketoglutarate,
with subsequent conversion to oxaloacetate. Citric
acid cycle intermediates are involved in metabolism
of amino acids, fatty acids, nucleotides, and sugars.
24. Oxaloacetate is important in many metabolic
pathways. It can be converted to glucose in
gluconeogenesis, to aspartate by transamination, and
to citrate in the citric acid cycle. Both oxaloacetate
and alpha
ketoglutarate are
important
anaplerotic
intermediates.
Biologists will not stand by

And bid all the gophers goodbye
Were all interlinked
So if they go extinct
The hole ecosystem might die
301
In the oce it was not a thrill

When my dentist had three teeth to fill
Things couldnt get worse
When he said to the nurse
All right, I think you know the drill
26. Because of
decarboxylation in the
citric acid cycle, no
net synthesis of
glucose can occur via
the citric acid cycle. On the other hand, plants and bacteria
that have the enzymes of the glyoxylate cycle are able to form
glucose in net amounts from acetyl-CoA because they can
bypass the decarboxylation reactions of the citric acid cycle
and convert acetyl-CoA into useful material.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
302
Key Points - Lipids and Membranes

1. Membranes are composed of a lipid bilayer and other
molecules, such as cholesterol. The lipid bilayer contains two
main categories of molecules - glycerophospholipids and
sphingolipids.
2. Glycerophosphlipids contain glycerol, phosphate and one or
more fatty acids. The fatty acids in the molecules of the lipid
bilayer are either saturated (no double bonds) or unsaturated
(contain one or more double bonds). Double bonds in biological
fatty acids are almost exclusively in the cis configuration,
resulting in a bent shape for unsaturated fatty acids.
3. Glycerophospholipids (also called phosphoglycerides) are
related to fats in having a glycerol backbone and two fatty
acids, but they dier from fat in having a phosphate located in
position #3 of the glycerol. Naming of glycerophospholipids is
generally as "phosphatidyl-X" where X is the name of the
molecule attached to the phosphate. Examples include
phosphatidylserine, etc.
4. If there is no other molecule attached to the phosphate on the
glycerophospholipids described above in #3, you have
phosphatidic acid.
Phosphatidic acid is an
important intermediate in
synthesis of phosphatidyl
lipids, as well as fats.
At the bookstore I asked timidly

For some help to get phobia-free
The clerk pointed to
A brand new review
But I fear that it may not help me
5. Sphingolipids are molecules related to glycerophospholipids

that are based on sphingosine. Sphingomyelin is a component
of the myelin sheath of nerve cells. Sphingolipids containing a
303
single sugar are called cerebrosides and sphingolipids
8. In addition to glycerophospholipids and sphingolipids,
containing a complex carbohydrate moiety are called
membranes contain proteins, glycoproteins, and glyolipids.
gangliosides. Sphingolipids are prominent components of the
Four types of membrane proteins are integral (protein projects
membranes of nerves
through both sides of the
and brain tissue.
membrane), peripheral
(protein projects into only
6. Steroids are lipids that
one side of the membrane),
are not derived from fatty
anchored (protein is linked
acids. In animals,
to a molecule embedded in
steroids are derived from
the lipid bilayer, or
cholesterol. Cholesterol
associated (protein
is found in the membrane
associates by hydrogen
of cells and is important
bonding with an integral
for membrane stability.
membrane protein.
Cholesterol is prominent
in brain membranes - up
9.Integral membrane
to 14% of the dry weight
proteins are dicult to
of brain.
remove from membranes,

but peripheral and
7. A lipid bilayer has a
associated membrane
polar exterior facing
proteins are not.
water and a non-polar

interior. As such, the
membrane provides a
10. Membranes provide a

barrier between the cell and
good barrier to both polar and non-polar substances. In
the external environment. Membranes provide a barrier to
contrast to the lipid bilayer, fatty acids aggregate into a micelle.
passage of many molecules, including molecules, such as

sugar that the cell could use for food.
304
11. Integral membrane proteins span into and/or across the
artificial systems are called liposomes. Liposomes can carry
plasma membrane and thus must have both hydrophilic and
substances and when the membrane of the liposome fuses
hydrophobic portions that interact appropriately with the same
with a cell membrane, the contents of the liposome are
portions of the plasma membrane. By contrast, peripheral
delivered into the cell. This is a useful way of getting
membrane proteins are
compounds into cells that
found in association with
are not easily transported
membranes, such as by
across the cell membrane
interacting with an
in other ways.
integral membrane
14. Membrane-spanning
protein. Anchored
proteins have alternating
membrane proteins are
regions of non-polar
proteins attached to a
membrane crossing regions
molecule (like a fatty
interrupted by polar short
acid). The molecule is
sections joining the non-
embedded in the lipid
polar regions.
bilayer and thus the
Consequently, one can use
protein is anchored to it.
a computer to examine the
12. Bacteriorhodopsin is an
amino acid sequence of a
integral membrane
protein and predict
protein that uses light,
reasonably accurately if the
chemistry, and
protein is a membrane
mechanics to move
protons across a
membrane barrier.
13. One can assemble artificial lipid bilayers containing
compounds as a means of delivering materials into cells. These
protein or not.
15. Cellular membranes are
somewhat fluid in nature. The fluidity of membranes is related
to their composition - shorter, more unsaturated fatty acids
make for membranes that retain fluidity at lower temperatures
305
compared to longer, saturated fatty acids. Fish membranes, for

example are full of unsaturated and polyunsaturated fatty
acids, which makes sense because fish membranes must be
fluid at fairly low temperatures.
16. Biological membranes can be in a fluid or a more solid state.
The midpoint of the conversion between the solid and the fluid
state is referred to as the Tm. Cholesterol is often found in
membranes. Though it does not change the Tm of a membrane
cholesterol does widen the range of the transition temperature
between solid and fluid state.
17. Unsaturated fatty acids in glycerophospholipids and
sphingolipids tend to lower the Tm of a membrane. Saturated
Jump to Chapter
fatty acids tend to raise the Tm. Shorter fatty acids also lower
Tm, but longer ones raise it.
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
18. The Fluid Mosaic Model explains the fluidity of cellular

membranes.
19. Not all molecules move into a cell through specific protein
receptors. An example of such an exception is cholesterol,
which enters cells via LDLs that attach to a receptor on the
cell's surface. The entire LDL with the cholesterol is taken into
the cell in a process called endocytosis.
306
Key Points - Membrane Transport

1. Diusion is a process in solutions where molecules move from
a high concentration to a low concentration. Active transport
occurs when a least one molecule is moved across a
membrane from a low to a higher
concentration. This takes energy.
2. We break transport across membranes into
two main categories - 1) passive transport
molecules in opposite directions across a membrane are called

antiports. Pumps are called electroneutral if their action does
not result in a net change in charge and electrogenic if their
action changes the charge across the membrane as a result of
their action.
The baker of bread has succeeded

His quotas today all exceeded
The reason hes blessed
You probably guessed
He had everything that he kneaded
(diusion driven, so materials move only from

high concentration to lower concentration and don't require
outside energy), and 2) active transport (an energy-requiring
process that moves at least one molecule from a low
concentration to a higher concentration - this is counter to
3. Active transport moves at least one molecule in the opposite

direction of where diusion would operate (that is, active
transport moves at least one molecule from a low
well (see below). The term 'pump' is used to

describe the protein component of an
lets glucose diuse into cells. No energy other

than that provided by the process of diusion is
required for that particular transporter. Other
glucose transporters in other cells are active in that they use

energy to move glucose against a concentration gradient.
6. An example of a passive transport system is a glucose
cells. No energy is required for that particular transporter. Other
glucose transporters in other cells are active in that they use
energy to move glucose against a concentration gradient.
7. P-type ATP-using transport systems use phosphoaspartate as
concentration to a higher concentration).
transport, but there are other sources, as
glucose transporter in blood cells that simply
transporter in blood cells that simply lets glucose diuse into
simple diusion).
4. ATP is a primary energy source for active
5.An example of a passive transport system is a
The heir didnt think it was great

With his uncle now at heavens gate
He got zero stocks
Just a hundred old clocks
So hes now winding up his estate
active transport system. Pumps that move
a covalent intermediate in their mechanism of

action.
8.The mechanism of transport of the Ca/
ATPase pump includes binding of ATP and the
relevant ions (calcium, in this case), transfer of
two molecules in the same direction across a membrane are
phosphate from the ATP to the protein (making
called symports (or synports), whereas pumps that move two
phosphorylaspartate), conformational change in the protein

307
causing movement of the ions across the membrane,

hydrolysis of the phosphate from an aspartic acid side
chain in the protein, a second conformational change
to bring the protein back to its original state.
9. Another P type ATPase is the Na+/K+ ATPase. The Na+/
K+ ATPase transports three sodiums out of the cell and
two potassiums in during each cycle. This is an
electrogenic transport mechanism and uses hydrolysis
of ATP to drive the process. Movement of Na+ and K+
is essential for the cell being able to maintain osmotic
balance. The Na+/K+ ATPase is called an antiport
because it moves molecules in opposite directions.
10. Another class of transporter proteins that use ATP to
move molecules is the ABC transporters. An example
is the Multidrug Resistance Protein that is involved in
the resistance of cancer cells to chemotherapy agents.
They act by binding the compound first. This causes a
conformational change in the protein that allows ATP

to bind. Binding of ATP causes the protein to 'evert' (move its
opening from one side of the membrane to the other). This has
the eect of moving the bound compound to the outside of the
cell. After this happens, ATP is hydrolyzed to change the
protein to evert again and change back to its original
conformation (opening facing inwards).
11. The Na+/Ca++ exchange pump is a secondary transporter. It

uses movement of Na+ in to cells to be a driving force for
pumping Ca++ out. Remember than Ca++ stimulates muscular
contraction. If Ca++ is not pumped OUT, its concentration in
muscle cells remains high, stimulating contraction.
Digitoxigenin is a poisonous compound from foxglove that
binds the Na+/K+ ATPase, preventing development of a Na+
gradient. As a consequence, digitoxigenin increases Ca++
308
concentration, since Ca++ pumping requires a Na+ gradient.
14. The first step in nerve transmission involves opening of Na+
Digitoxigenin is used as a heart stimulant, but is a dangerous
gates. These allow Na+ to diuse into the cell, since Na+
poison that must be properly used.
concentration is higher outside of cells than inside. Movement
12. Proteins that move more than one chemical in the same
direction across a
compensate for the voltage
symports (synports).
change, the K+ gates open
Those that move them in
and Na+ gates close,
opposite directions are
allowing K+ to flow out of
called antiports. If a net
the cell. This results in an
charge dierence arises
overcompensation of the
as a result of the
voltage. The K+ gates close
movement, the system is
and the region where the
referred to as
original movement of ions
electrogenic. If no charge
occurred recovers. During
dierence arises, they
this time, no nerve signal
are called electroneutral.
can be transmitted at that
13. Nerve cells use the
point.
gradient of Na+ and K+
15. The nerve signal is
built up by the Na+/K+
transmitted as a
ATPase pump to transmit
transmission, special
"gates" open and close to allow Na+ to diuse into nerve cells
and
K+
electrical potential of the

cell near the Na+ gate. To
membrane are called
signals. In nerve
of the positively charged sodium ion causes a change in the
to diuse out of nerve cells.
consequence of the initial

movement of Na+ into the
cell. Before it can be pumped out, some of the sodium diuses
down to the next Na+ gate and the change in the voltage
environment causes it to open and trigger the same events as
309
17. Channels (gates) are made by protein molecules in

the membranes of cells. Channels are generally very
specific for what they will allow to pass through them.
Glucose channels, for example are fairly specific for
glucose. Sodium and potassium channels are very
specific for each respective ion.
18. Ion specificity is accomplished by two mechanisms.
The first is physical. If an ion is too big to fit in a channel,
it is excluded. This is the case of the sodium channel,
which excludes potassium ions because they are too big.
19. The second mechanism of specificity is energy. An
example is the potassium channel, which excludes
sodium ions. In this case, the channel allows larger ions
(potassium) to pass through, but blocks smaller ions, like
sodium ions.
occurred in the last step. Thus, the signal moves from one
20. The mechanism of exclusion of the potassium

channel relates to the energies of solvation of each ion.
For potassium ions, the energy of desolvation of the ion
junction to another to another, ultimately arriving at the end of
as it enters the channel is overcome by the energy of
the axon.
resolvation as it enters the channel. Thus, entry of potassium
16. Tetrodotoxin is a neurotoxin because it inhibits the action of

nerve cells. It is found in the puer fish and it blocks the Na+
gates.
ions is energetically favored. This is due to the geometry of the

potassium channel closely matching the dimensions of the
potassium ion.
21. When sodium ions try to enter the channel, their energy of
desolvation is greater than is realized by their resolvation in the
310
encapsulated in vesicles (synaptic vesicles) that are

released from the nerve cell carrying the signal
(presynaptic membrane) to the adjacent nerve cell
(postsynaptic membrane). Acetylcholine, for example, is
released in presynaptic vesicles and when the contents
(acetylcholine) bind receptors on the postsynaptic
membrane, channels open to allow sodium and
potassium ions to move as before, causing the next nerve
cell to start an action potential.
channel. Thus, they do not enter. Their energy of resolvation
Jump to Chapter
not as favorable due to their ion sizes not matching the

dimensions of the potassium channel.
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
22. After the nerve has "fired" the gradient must be restored and
this is the job again of the Na+/K+ ATPase.
23. After a nerve "signal" has moved along the entire length of a
nerve cell, it must move to the adjacent cell. This requires a
neurotransmitter. Neurotransmitters are small molecules
311
Key Points - Mitochondria and

Energy
1. Mitochondria are the locations in cells where
oxidation/reduction and ATP synthesis occurs,
along with metabolism. Mitochondria have
distinctive structural features, including an inner
membrane that is impermeable to protons,
infoldings of the inner membrane called cristae,
an outer membrane that is not very impermeable,
an intermembrane space between the inner and
outer membrane, and the matrix. This last
"structure" is simply the fluid in the inner
mitochondrion and it is here where the enzymes
of the citric acid cycle and fatty acid oxidation are
found.
2. Remember that for every oxidation, there is an
equal and and opposite reduction. Loss of

electrons by one molecule means gain of them by another one.
Oxidation is a process that involves the loss of electrons.
Reduction is a process that involves the gain of electrons.
3. Electrons are
carried to the
electron transport
system in the
mitochondria by
At the magic show people gave stares

To the man doing tricks with a flare
Some were appalled
Cuz they thought hed go bald
From constantly pulling out hares
NADH and FADH2.

4. Mitochondria are the site of electron transport and oxidative
phosphorylation.
5. Electrons from NADH enter the electron transport system
through complex I.
6. Electrons from FADH2 enter the electron transport system
through complex II.
312
The man in the dark walked unsighted

Dead batteries seemingly blighted
He just hit a wall
He could not see at all
His flashlight was simply delighted
7. Coenzyme Q (CoQ)
CoQ (QH2 and Q) to Complex III. QH2 has two electrons and
accepts a pair of
two protons. Q has neither.
electrons from either

complex I or complex
II and passes
electrons singly to cytochrome c through complex III. Thus,

coenzyme Q acts as a "trac cop" for electrons.
8. The sequence of electrons passing from coenzyme Q is as
follows: Coenzyme Q -> Complex III -> Cytochrome c ->
Complex IV -> oxygen (to form water)
9. Oxygen is thus the terminal electron
acceptor and is a limiting compound during
periods of heavy exercise.
13. After QH2 and Q bind, QH2 sends one electron to Q, creating
Q- and one electron to cytochrome C. The two protons QH2
was carrying are expelled into the intermembrane space. This
converts QH2 to Q. Both cytochrome C and Q leave the
complex, but Q- remains behind.
14. Next, another QH2 and another cytochrome C binds to
Complex III. QH2 sends one electron to Q-,
The award ceremony revealed

When the big envelope was unsealed
The scarecrow got buzz
The reason because
Hes just out standing in his field
10. If oxygen is not available, electrons will

NOT pass through the electron transport system and NADH
creating Q-2 and one electron to cytochrome

C. It also expels its two protons to the
intermembrane space and becomes Q. Then
Q-2 extracts two protons from the matrix and
becomes QH2. Last, cytochrome C, QH2, and
Q all leave the complex.
and FADH2 will not be reoxidized. For these reasons, the citric
15. Electron transfer through complex IV occurs one electron at a
acid cycle will not run either. This is part of metabolic control.
time (since one electron arrives at a time from cytochrome c).
11. Several compounds inhibit electron transport - rotenone (an

insecticide) and amytal block all action of Complex I. Antimycin
A blocks all action of Complex III. Cyanide, azide, and carbon
monoxide block all action of complex IV.
12. Movement of electrons through Complex III is known as the Q
cycle. This cycle begins with the binding of two molecules of
Interruption of electron flow can result in production of reactive

oxygen species. Cellular enzymes, such as superoxide
dismutase and catalase (see below) help to deactivate reactive
oxygens.
16. In electron
flow through
complex IV, the
first electron is
The overweight man couldnt bear

Each diet led him to despair
So he raced to the door
Of the nearest paint store
When he heard you could get thinner there
313
transferred to copper and the second one is transferred to iron.
consequence, the proton numbers in the matrix decrease by 8
Oxygen then binds to the iron first, followed by formation of a
during the process. The proton numbers outside the
peroxide bridge between the iron and copper atoms. Addition
mitochondrion INCREASE by four in the process, so the net
of a third electron (to the oxygen on the copper) and binding of
dierence is 12 protons just for movement through complexIV.
a proton from the matrix causes the O-O bond to be cleaved. A
18. ATP is created in
fourth electron then
oxidative phosphorylation
reduces the oxygen
by the movement of
on the iron and a
protons back into the
proton binds from the
mitochondrial matrix
matrix as well. Last,
through complex V (also
two protons from the
called the ATP synthase).
matrix bind to the

hydroxyls on the iron
19. Two essential functions
and copper, forming
of electron transport - 1.
two water molecules,
Pump protons out of
which are released
mitochondrial matrix and
and the cycle is
2. Reoxidize NADH and
complete.
FADH2 to NAD and FAD,

respectively. In healthy,
17. During electron
normal cells, oxidative
movement through
phosphorylation is tightly
Complex IV, four

protons are taken
coupled to electron
from the matrix and

combined with oxygen to form two water molecules. In
addition, four other protons are taken from the matrix by the
transport. Stopping
electron transport will ultimately stop oxidative phosphorylation
in tightly coupled mitochondria.
complex and pumped outside the mitochondrial matrix. As a

314
Theres one indisputable fact

The trains engineer should react
Ignoring transponders
As his mind slowly wanders
Is just how he got so side tracked
20. The
formation of ATP from ADP and Pi. Conversions in the process
chemiosmotic
occur as follows:
hypothesis, originally
O goes to L
proposed by Peter
L goes to T
Mitchell, explains
T goes to O
how mitochondria make ATP in oxidative phosphorylation.

Important aspects of it include:
a. Intact inner mitochondrial membrane
b. Electron transport creates a proton gradient
c. ATP is made by movement of protons back into the
mitochondria
21. Tight coupling of electron transport and oxidative
24. Mitochondria which are "tightly coupled" have intact

membranes AND the only way protons get back into the matrix
is by passing through Complex V. If you poke a hole in the
membrane (using DNP or an uncoupling protein, such as found
in brown fat), protons can leak back in without making ATP.
This has the eect of generating heat AND burning up energy
sources (like glucose and fat). DNP is a dangerous compound
phosphorylation requires at a practical level that the
that killed
mitochondrial inner membrane remain impermeable to protons,
tried to
except for those that enter via the ATP synthase and result in
lose
ATP production.
22. The ATP synthase consists of a turbine-like structure
containing 3 sites called Loose (L), Tight (T), and Open (O).
Functions of these forms include
L - Holds ADP and Pi in preparation for ATP formation
T - Causes ADP and Pi to join and form ATP
O - Releases ATP formed in T and binds ADP + Pi
23. Movement of protons through the ATP synthase cases
rotation/conformational changes in the complex that result in
25. When
people who
Near a cow everyone should beware
Of the gas they release everywhere
Its the reason they say
On almost any day
That a farm smells just like dairy air
use it to
weight.
mitochondria are tightly coupled, metabolic (respiratory) control

exists. This means that electron transport will stop if oxidative
phosphorylation stops, since the protons don't come back in,
so the proton gradient gets very high, stopping the pumping of
protons. When electron transport stops, NADH accumulates
and the citric acid stops. Conversely, if one stops electron
transport with cyanide, oxidative phosphorylation will stop
315
In political science Ms Parks

Spoke her mind and created some sparks
She said that her grade
Was surely waylaid
Because she had such lousy Marx
shortly because
Electrons make it into
the proton
the mitochondria by
gradient is lost
means of shuttles.
when no protons
Insect muscles use a
are pumped.
glycerol-3phosphate/
26. When mitochondria are uncoupled (by poking a hole in them

to let protons leak back into the matrix without passing through
Complex V), electron transport is no longer limited by oxidative
phosphorylation and runs amok. That is why heat
In heaven an organized plan

Uses numbers wherever they can
Mr. Thirteen said Im
Forever a prime.
A most indivisible man
DHAP shuttle that transfers electrons from NADH ultimately to

FADH2. Mammalian systems, by contrast, employ a malate/
aspartate system that converts oxaloacetate to malate (carrier
of electrons) which gets transported by a transport protein.
is generated. Protons are pumped, but they fall

back in through the hole in the mitochondrial inner
membrane. No ATP is made. NADH is rapidly
converted to NAD+, so the citric acid cycle and
other pathways run rapidly.
27. The compound 2,4 DNP (dinitrophenol) was
marketed as a miracle diet drug about a century
ago. It kills because it destroys the proton gradient
of mitochondria without allowing synthesis of ATP.
28. Things that aect respiration are ADP (necessary
for the Complex V to function), oxygen (necessary
for electron transport to function), NADH (source of
electrons for electron transport), and NAD+
(needed for citric acid cycle).
29. NADH cannot cross the inner membrane of the
mitochondrion, as there is no protein to move it.
316
Once inside the matrix, malate transfers electrons to NAD+,

creating NADH and oxaloacetate.
30. Superoxides are reactive oxygen species generated as a
byproduct of electron transport (among other things). They
react with whatever they encounter, so can be damaging to
DNA, proteins, etc. Superoxide dismutase is an enzyme
employing a ping-pong mechanism that works in two steps to
destroy two superoxide molecules, while producing hydrogen
peroxide.
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1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
317
Key Points - Photosynthesis

1. Photosynthesis is a process in plants and some bacteria that
use energy from the light of the sun to synthesize glucose using
carbon dioxide and water as starting reagents. It accomplishes
this in a multistep process that is divided into two phases,
called the light reactions (require light) and the dark reactions
(don't require light).
2. Photosynthesis occurs in plants in organelles called
chloroplasts. The thylakoid disks of the chloroplast are the sites
where the light reactions of photosynthesis and the stroma are
the locations of the dark reactions.
3. Molecules involved
in the capture of light
energy are known as
the chlorophylls.
These molecules
Perhaps it was all the demands

Disrupting the cannibals plans
Or maybe the stress
That he tried to suppress
That made him go throw up his hands
contain a porphyrin ring (like hemoglobin) with a magnesium

ion at the center (instead of the iron molecule found in
hemoglobin).
4. In the light reactions of photosynthesis, 1) electrons are
removed from water (producing oxygen); 2) ATP is produced by
the process of photophosphorylation as electrons pass through
the membrane of the thylakoids; and 3) NADPH is produced
from NADP+ in the final reduction reaction. NADP+ is therefore
the terminal electron acceptor in photosynthesis, whereas
water is the electron source for the process.
5. Movement of electrons through the electron transport chain in
the thylakoid membrane causes protons to be pumped INTO
the thylakoid. This creates a proton gradient (higher proton
concentration in the thylakoid than the stroma). Protons in the
thylakoid move outside through a proton translocating ATP
synthase (PTAS) complex (same general structure as the
mushroom-like complex with the same function in
mitochondrial membranes). As protons move through the
PTAS, ATP is generated from ADP (photophosphorylation).
318
It is true that Cher got a small shiner

When she hurried to see her designer
Of her speed police said
It wasnt widespread
Thus was the stars inertia minor
6. There are two
excited electrons are passed to ferredoxin and ultimately to
photosystems (I and II)
NADP+, creating NADPH, the final product of the light
that act together to
reactions.
produce the reactions of

photosynthesis.
Photosystem II is involved in the first set of reactions. Here
9. Quinones are molecules that help carry electrons in the

thylakoid membrane (and mitochondrial membrane too).
electrons from the first porphyrin ring complex are excited by
10. The dark reactions of photosynthesis are where the glucose is
light. As these electrons are passed to the electron transport
synthesized. Thus, we can think of the light reactions as the
chain of the membrane, the ring extracts electrons from water,
ones where the energy necessary for making glucose is stored
creating oxygen.
7. Movement of electrons through the electron
transport chain in the thylakoid membrane causes
protons to be pumped INTO the thylakoid. This
creates a proton gradient (higher proton
concentration in the thylakoid than the stroma).
Protons in the thylakoid move outside through a
proton translocating ATP synthase (PTAS) complex
(same general structure as the mushroom-like
complex with the same function in mitochondrial
membranes). As protons move through the PTAS,
ATP is generated from ADP
(photophosphorylation).
8. Electrons released from photosystem II eventually
reach photosystem I where they are excited by
sunlight of a dierent wavelength. These newly
319
In Russia the pundits expound

Many theories wheneer theyre around
Theyd recover much faster
From World War disaster
If they hadnt kept Stalin around
up (charging a
decarboxylated in another part of the plant, releasing carbon
battery) and the
dioxide once again. This newly released carbon dioxide enters
dark reactions as
the Calvin cycle (same reactions here as C3 plants). This unique
the ones that use
delivery system allows C4 plants to eciently deliver CO2 to
energy from the light
where it is used and allows them to avoid water loss. It may
reactions to store that energy in another form - glucose.
also allow them to be more ecient in that the rubisco reaction

is occurring in the plant at a location where the oxygen
11. The dark reactions of photosynthesis are also known as the
concentration is lower than it is where the rubisco reaction
Calvin cycle in honor of their discoverer, Melvin Calvin.

12. In the dark reactions, carbon dioxide is removed from the
atmosphere in a process called fixation. Carbon dioxide is first
covalently attached to ribulose1,5bisphosphate (Ru1,5BP) to
form a six carbon intermediate that immediately breaks down
to form two molecules of 3-phosphoglycerate (3-PG) for each
molecule of carbon dioxide combined with Ru1,5BP. This
occurs in C3 plants.
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reaction is catalyzed by the enzyme with the acronym RuBisco.

13. 3-PG is an intermediate in glycolysis and gluconeogenesis.
Phosphorylation and reduction of it leads to glyceraldehyde-3phosphate (G3P).
14. The pathway described to this point is that taken by plants
known as C3 plants, by virtue of the fact that the first molecule
made after fixation of carbon dioxide has three carbons.
Another group of plants, known as C4 plants, fixes carbon
dioxide to PEP and forms (surprise!) a four carbon molecule,
oxaloacetate. Ultimately, this four carbon molecule is
320
Key Points - Lipid and Steroid

Metabolism
1. Phosphatidic acid is an immediate precursor of CDPdiacylglycerol, which is a precursor of the various
glycerophospholipids. CTP combines with phosphatidic acid to
yield a pyrophosphate and CDP-Diacylglycerol. Activation by
CDP yields a high energy activated intermediate that can be
readily converted to phosphatidyl glycerophospholipids.
2. From CDP-diacylglycerol, phosphatidyl serine can be made, as

can phosphatidyl ethanolamine and phosphatidyl choline.
Synthesis of phosphatidyl choline from phosphatidyl
ethanolamine requires methyl groups donated by S-AdenoysylMethionine (SAM). Loss of the methyl groups by SAM yields SAdenosyl-Homocysteine.
3. Phosphatidyl ethanolamine (and phosphatidyl choline - derived
from phosphatidyl ethanolamine) can both be made
independently of phosphatidic acid biosynthesis. For this
pathway, CDP-ethanolamine is the activated
intermediate and the phosphoethanolamine of it is
added to diacylglycerol to form
phosphatidylethanolamine. Phosphatidyl choline
can be made by the same methylation scheme in
point 4.
4.Sphingolipids are synthesized beginning with
palmitoyl-CoA and serine. Addition of a fatty acid
to the amine group yields a ceramide. Addition of
sugars to a ceramide yields either a cerebroside
(single sugar added) or a ganglioside (complex
sugar added).
5.Deficiencies in enzymes that degrade
sphingolipids (particularly cerebrosides and
gangliosides) are linked to neural disorders. One
such disorder is Tay-Sachs disease.

321
6. Cholesterol is an important component of membranes,
11. Reaction of isopentenyl pyrophosphate with dimethylallyl
particularly in the brain. Cholesterol can be synthesized totally
pyrophosphate yields a 10-carbon intermediate, geranyl
from acetyl-CoA.
pyrophosphate.
7. Steroids include all compounds

synthesized from cholesterol. This
includes steroid hormones, vitamin D,
bile acids, and other related
compounds.
The haberdashers remark was misread

When he looked towards his friends and then said
You guys should all know
That to be status quo
I have to go right on a head
12. Reaction of geranyl pyrophosphate

with isopentenyl pyrophosphate yields a
15 carbon intermediate - farnesyl
pyrophosphate.
13. Reaction of two farnesyl
8. Isoprenes (also called isoprenoids) include the steroids and

other compounds, such as Vitamin A and E that are also made
from isoprene units.
pyrophosphates yields a 30 carbon intermediate - squalene.

14. Further reaction of squalene yields a cyclic intermediate,
lanosterol. Synthesis of cholesterol from lanosterol requires an
9. Isoprene - 5 carbon units - 1) isopentenyl pyrophosphate and
additional 19 steps. From cholesterol, one can synthesize the
dimethylallyl pyrophosphate. Each is made from acetyl-CoA
bile salts, which are useful in solubilizing fat in the diet.
molecules. Synthesis of isopentenyl pyrophosphate and
Cholesterol also leads to synthesis of the steroid hormones.
dimethylallyl pyrophosphate comes from mevalonate, which, in

turn, comes from HMG-CoA, so
isoprenoid biosynthesis overlaps partly
with ketone body synthesis.
10. The most important enzyme in
15. Cholesterol in the body is either there as
The writing instructor would show

All her students the writing rules though
She seemed quite the fool
When she read them the rule
Double negatives are a no-no
cholesterol biosynthesis is HMG-CoA

reductase. It converts HMG-CoA to mevalonate and is the
primary regulatory enzyme in cholesterol biosynthesis.
Cholesterol is a feedback inhibitor of the enzyme.
a result of 1) synthesis; 2) diet; or 3) storage/

recycling.
16. In the digestive system, bile acids and
mechanical agitation of the stomach help
emulsify fats.
17. In the intestines, hydrolysis of fatty acids from fats by lipases

(pancreatic lipase, for example), yield soapy like substances
that help emulsify lipids for transport across the intestinal wall.
322
18. After movement across the intestine, lipids are packaged in
20. The liver is
chylomicrons. They move through the lymph system into the
involved in sensing
capillaries where they get stuck.
the body's need for
19. Action of lipoprotein lipase in the capillaries removes some of

the fat from the chylomicrons and they shrink in size and exit
the capillaries and move to the liver. They are taken up there
and released, when needed as VLDLs. These travel the blood
system and get converted to IDLs and ultimately LDLs. Note
that cholesterol ONLY gets into cells via receptor-mediated
endocytosis of the LDL at the target cells.
lipids via LDL

receptors. If it
It is true that the students decision

Getting glasses helped him to envision
Mathematical ties
And soon realize
They helped him two ways with division
senses lipids are needed, VLDLs are packaged by the liver and
released into the bloodstream. These get degraded by lipases
and other enzymes to IDLs and LDLs. If the liver LDL receptor
cannot detect LDLs in the blood, it continues to release more
lipids in lipoprotein complexes, thus elevating the LDL
concentration.
21. Defects in liver LDL receptors, such as found in familial
hypercholesterolemia (a genetic disease) are one cause of
high blood cholesterol. Other factors to consider include
dietary cholesterol, cholesterol synthesis rate, and eciency
of recycling cholesterol. Statins are drugs that inhibit HMGCoA reductase and reduce cholesterol by inhibiting its
synthesis.
22. LDLs are called bad cholesterol because in high amounts,
they can lead to formation of atherosclerotic plaques. These
arise due to oxidative damage of unsaturated fatty acids in
the LDL by reactive oxygen species. The immune system
attacks these damaged LDLs and the resulting complex can
lead to the formation of foam cells (rich in cholesterol) and
finally a physical block to the flow of blood (plaque).
323
23. Factors increasing LDLs include smoking, obesity, and

saturated fats in the diet.
and moves to the Golgi Complex where a serine protease

cleaves SREBP-A to release SREBP-B. Next, SREBP-B
24. Opposing the LDLs are the HDLs (good cholesterol). They are
associated with removing cholesterol from the bloodstream
and giving feedback to the liver to reduce the output of VLDLs.
High levels of HDLs correlate with reduced incidences of
atherosclerosis. Factors increasing HDLs include exercise and
factors decreasing them include obesity and smoking.
migrates in the membrane to a metalloprotease, which clips the

DNA binding region to free SREBP-C (a transcription factor)
from the membrane-bound portion. The SREBP-C has a DNA
binding region then travels through the cytoplasm and enters
the nucleus, where it binds to the promoter region (called SRE)
in front of the HMG-CoA Reductase. This causes HMG-CoA
25. If a person has high cholesterol, the first

approach is to see if levels can be brought down
by dietary changes. If they cannot, then drugs are
used to stop 1) endogenous synthesis and/or 2)
recycling.
26. SREBP (Steroid response element binding
protein) is a protein that plays a role in controlling
whether or not HMG-CoA reductase is made.
When cholesterol is abundant, SREBP is found in
the membrane of the endoplasmic reticulum
linked to another protein called SCAP through the
regulatory (REG) region of SREBP. When
cholesterol is abundant, SCAP-SREBP-A is held in
the endoplasmic reticulum's membrane by a
protein called INSIG
27. When cholesterol levels in the cell fall, the SCAP/
SREBA complex loses its connection to INSIG
324
Reductase to be synthesized and cholesterol synthesis can

then begin.
enzyme known as aromatase. Since some tumors are
28. HMG-CoA reductase can also be broken down when

cholesterol is abundant. This occurs through action of another
INSIG. This INSIG carries an enzyme to
ubiquitinylate (put ubiquitin onto) the HMGCoA reductase. Ubiquitin is a "flag" to the
cell to digest the protein it is attached to
with a protease, thus destroying it.
32. Conversion of androgens to estrogens requires action of an

stimulated by estrogens, such as estradiol, inhibition of them
by aromatase inhibitors is a strategy of some chemotherapies.
If a chicken should ere get the notion

Crossing roads by its own locomotion
Theres no need to ask why
That the birdie passed by
It is simply some poultry in motion
29. Phosphorylation of HMG-CoA reductase is also regulated by

feedback inhibition (allosteric) and phosphorylation (covalent
modification). Thus, HMG-CoA reductase can be regulated in
multiple ways - synthesis, degradation, phosphorylation,
allosterism.
30. Cholesterol is a precursor of the bile acids that are important
33. Vitamin D is derived from cholesterol

(ultimately). A reaction converting 7dehydrocholesterol to Previtamin D3 requires
ultraviolet light. Conversion of Previtamin D3 to
Vitamin D3 occurs spontaneously. Conversion
of Vitamin D3 to calcitrol requires an enzyme
and the enzyme is regulated to control how much calcitrol is

made. Calcitrol is the active form of vitamin D. Vitamin D is
involved in regulating uptake of calcium and phosphorus,
which are important for healthy bones. It may also play roles in
mental health and has been shown to have anti-cancer
properties.
in the digestive system for solubilizing dietary fat. Two

examples include glycocholate and taurocholate.
31. Cholesterol is also a precursor of the steroid hormones. There
are five groups of steroid hormones. These are all derived from
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pregnenolone and include androgens (male sex hormones),

estrogens (female sex hormones,which are derived from male
sex hormones), mineralocorticoids (regulation Na/K and blood
pressure), glucocorticoids (fat/protein degradation & swelling/
inflammation), and progestagens (maintenance of pregnancy) .
325
Key Points - Fatty Acid Metabolism

1. In the digestive system, bile acids and mechanical agitation of
the stomach help emulsify fats.
6. Fats are broken down to fatty acids and glycerol by enzymes

known as lipases. One of these, hormone sensitive
triacylglycerol lipase, is the only regulated enzyme of fat or fatty
acid breakdown. It is located in fat-storing cells called
2. In the intestines, hydrolysis of fatty acids from fats by lipases

(pancreatic lipase), yield
adipocytes.
7.Triacylglycerol lipase action
soapy like substances that
cleaves the first fatty acid o
help emulsify lipids for
of a fat and this step is
transport across the
necessary before the other
intestinal wall. On the other
lipase can act to remove the
side of the wall, they are
other fatty acids from a fat.
reassembled back into fats

and these are packaged in
8. Glycerol, is the only part of
the chylomicrons.
a fat that can be made into

glucose (via
3. Breakdown of fats occurs
gluconeogenesis). Fatty acids
by action of enzymes
travel in the bloodstream
known as lipases and these
carried by serum albumin.
may be extracellular or
9.Fatty acid oxidation occurs
intracellular.
in the matrix of the
4. Cells that store fat are

known as adipocytes.
mitochondrion. In the cell,
5. Fatty acid oxidation and
fatty acids are attached to

CoA and then at the
fatty acid synthesis are almost identically the chemical reverse
mitochondrion, the CoA is replaced by carnitine. Inside the
of each other.
mitochondrial matrix, the carnitine is replaced by CoA again.
326
10. Fatty acid oxidation proceeds in steps that mirror steps in the
13. The long chain acyl dehydrogenases are found in
citric acid cycle. These include dehydrogenation, hydration,
peroxisomes and this is where oxidation of long chain fatty
and oxidation. In the last step, a thiolytic cleavage releases
acids (longer than 16 carbons) begins (not in the mitochondrial
acetyl-CoA and a fatty acid two carbons shorter.
matrix). Oxidation here involves transfer of electrons to oxygen
11. The dehydrogenation and oxidation reactions yield reduced
to make hydrogen peroxide, instead of FADH2. Peroxisomal

fatty acid oxidation is therefore
electron carriers (FADH2
LESS ecient than mitochondrial
and NADH). The double
beta oxidation.
bond formed in the first

dehydrogenation reaction
14. The first step of oxidation
is in the trans form. The
generates a trans-intermediate
hydration yields a hydroxyl
plus FADH2. The second step
group on the third carbon
involves addition of water across
from CoA end in the "L"
the trans double bond to create an
configuration. Thiolytic
intermediate with a hydroxyl on
cleavage is catalyzed by
carbon 3 in the L configuration.
the enzyme called
The third step involves oxidation of
thiolase.
the hydroxyl intermediate to a

ketone on carbon 3. The last step
12. The first reaction of fatty

acid oxidation involves a
involves cleaving o of an acetyl-
CoA and production of a fatty acyl-
set of enzymes know as

acyl dehydrogenases. These are specific for fatty acids with
long, medium, or short chains. The medium chain acyl
dehydrogenase has been implicated in some instances of
sudden infant death syndrome (SIDS).
CoA with two fewer carbons. The

last step is catalyzed by the enzyme thiolase.
15. The reactions of beta oxidation up to the thiolase reaction
chemically mirror the reactions of the oxidation of succinate up
to oxaloacetate.
327
16. Seven cycles of beta oxidation of palmitoyl-CoA in the matrix

yield 8 acetyl-CoAs.
coenzyme group of vitamin B12. This coenzyme contains a
17. Oxidation of biologically occurring fatty acids with cis double

bonds requires two additional enzymes compared to oxidation
of saturated fatty acids. These enzymes are
enoyl-CoA-isomerase and 2,4-dienoyl-CoAreductase.
18. Enoyl-CoA-isomerase converts cis bonds
propionyl-CoA to succinyl-CoA requires an enzyme that uses a

cobalt atom that provide a means of generating the radical
necessary for the reaction.
Two computer nerds met in the thick

Of a programmers summer picnic
He preferred a track pad
She used tablets like mad
Too bad that the two didnt click
between carbons 3 and 4 to trans bonds

between carbons 2 and 3. Since beta oxidation normally has
trans bonded intermediates between carbons 2 and 3, this
21. Conversion of propionyl-CoA to succinylCoA requires three steps. The first is addition of
a carboxyl-group to the middle carbon in the
molecule. The overall process involves two
unusual isomerizations and movement of a
methyl group that utilizes the cobalt atom of vitamin B12.

22. Ketone bodies are produced by the body when glucose
enzyme is sucient for conversion of many naturally occurring
precursors are not available to make glucose. Examples of
fatty acids to be oxidized.
ketone bodies include acetoacetate and hydroxybutyrate.
19. 2,4-dienoyl-CoA reductase acts on intermediates that have

double bonds between carbons 2-3 and 4-5. It uses NADPH to
reduce the two double bonds to one
double bond and the resulting double
bond is placed in a cis configuration
between carbons 3-4. Enoyl-CoAisomerase then can convert this
Diabetics have problems with glucose metabolism and may

produce ketone bodies to provide energy to keep the brain
alive. One can detect this by the smell of acetone on their
Playing hookey one day sounded splendid

So no classes the truant attended
This excitement got trumped
After he bungie jumped
And discovered that he was suspended
intermediate to one with a trans double
breath. The thiolase reaction in ketone body

formation is the reversal of the same
reaction that occurs in fatty acid beta
oxidation.
23. Fatty acid biosynthesis occurs similarly
bond between carbons 2-3, thus allowing beta oxidation to
to beta-oxidation, though in reverse. Important distinctions are
continue.
noted below in a-f.
20. Oxidation of fatty acids with odd numbers of carbons yields a

final product of propionyl-CoA, not acetyl-CoA. Conversion of
328
c. NADPH is used to donate electrons in synthesis, but

the NAD+ and FAD are used to accept electrons in
oxidation in the mitochondrion.
d. A three carbon molecule, malonyl-ACP donates two
carbons to the growing fatty acid chain - a carbon
dioxide is lost in the process. Beta oxidations yield two
carbon acetyl-CoA units.
e. Synthesis of fatty acids longer than 16 carbons occurs
in endoplasmic reticulum or mitochondrion. Oxidation of
fatty acids longer than 16 carbons begins in
peroxisomes.
f. In fatty acid biosynthesis, a D-hydroxyl intermediate is
formed at carbon #3. In fatty acid oxidation, an Lhydroxyl intermediate is formed at carbon #3.
24. Acetyl-CoA carboxylase catalyzes the addition of a
carboxyl group to acetyl-CoA to form malonyl-CoA.

a. Fatty acid synthesis up to palmitate occurs in the
25. The enzymes of fatty acid synthesis apart from
cytoplasm, but beta oxidation occurs in mitochondrial
acetyl-CoA carboxylase are contained in a complex known as
matrix.
fatty acid synthase. We will refer to the fatty acid sythase
b. Fatty acids are built using an acyl carrier protein (ACP),

but beta oxidation uses CoA.
complex as the name of all of the enzyme for all of the

reactions after acetyl-CoA carboxylase.
26. Fatty acid synthase produces the saturated 16 carbon fatty
acid known as palmitate.
329
At the will reading late in the day

The executor stood up to say
It feels kind of funny
Handing out all this money
As part of a dead giveaway
27. Enzymes that produce

unsaturation in fatty acid
biosynthesis are called
desaturases. Desaturases
are found in the
endoplasmic reticulum.
citrate in the cytoplasm to create oxaloacetate and acetyl-CoA.

32. Prostaglandins are hormone-like compounds made from
arachidonic acid by action of an enzyme known as
prostaglandin synthase. There are several prostaglandin
synthases in the body. The reactions they catalyze are forming
cyclic oxygen-containing compounds (that's what
28. Essential fatty acids are those that must be provided in the
prostaglandins are), so the enzymes are also known as
diet of an organism, because the organism cannot synthesize
cyclooxygenases (or COX for short). The COX enzymes are
them.
known as COX-1 and COX-2.
29. In mammals, linoleic and linolenic acids are

essential fatty acids because these organisms
cannot make double bonds closer to the end
than the -9 position (oleic acid is a -9 fatty
acid). Thus, linoleic acid ( 9,12 double bonds
= omega 6 for an 18 carbon fatty acid) and
linolenic acid ( 9,12,15 double bonds =
omega 3 for an 18 carbon fatty acid) must be
provided in the diet of mammals.
30. Fatty acids longer than 16 carbons are
produced by action of enzymes called
elongases. These are found in the endoplasmic
reticulum and the mitochondrion.
31. Citrate acts as a shuttle to carry acetate to
the cytoplasm from the mitochondrion when
the citric acid cycle stops. Citrate lyase cleaves
330
33. Prostaglandins are involved in numerous physiological eects,

including control of vasodilation/constriction, uterine
contractions, aggregation/stickiness of platelets, inflammation/
The ink drop expressed grumpily
Her wish to escape and be free
I truly have been
Too long in the pen
This sentence is too long for me
pain, and
maintenance of
stomach tissue,
among others.
Inhibitors of COX
enzymes are called
COX inhibitors. Aspirin and ibuprofen are non-steroidal drugs

(called NSAIDs) that inhibit COX-1 and COX-2.
are involved in mucus production and bronchial constriction

and play important roles in asthma attacks.
37. Another class of molecules made from prostaglandins is the
thromboxanes. These molecules help to make platelets
"sticky", favoring aggregation. Thus, taking aspirin reduces
synthesis of prostaglandins, which in turn reduces amounts of
thromboxanes, which reduces stickiness of platelets, which
makes it harder for blood to clot. It is for this reason that
people prone to clotting problems are sometimes advised to
take aspirin daily.
34. Prostaglandins produced by COX-2 enzymes appear to have

no role in stomach maintenance, so inhibitors specific to them
Jump to Chapter
were sought. Examples include Celebrex and Vioxx, but they

may have negative side eects on the heart.
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
35. Arachidonic acid is produced from linoleic acid released from

glycerophospholipids by action of an enzyme known as
phospholipase A2 (PLA2). PLA2 can be inhibited by
corticosteroids, so action of these compounds can also
prevent prostaglandin formation indirectly. Corticosteroids are
important for treatment of severe inflammation or pain.
36. Leukotrienes can also be produced from arachidonic acid.
The pathway that leads to them does not involve cyclization, so
that pathway is called the linear pathway to distinguish it from
the cyclic pathway that leads to prostaglandins. Leukotrienes
331
Key Points - Nucleotide Metabolism

1. Nucleotides consist of a) sugar, b) nitrogenous base, and c)
phosphate.
2. Nucleosides consist of a a) sugar and b) nitrogenous base
3. The sugars of nucleosides and nucleotides are either ribose
primarily in ribonucleotides and rarely in DNA, but does appear

as a deoxyribonucleotide intermediate in thymidine
metabolism.
7. Nucleosides are named according to the base they contain.
Nucleosides containing purines are named by adding "os"
before the "ine." Thus, nucleosides containing guanine are
(found in ribonucleotides of RNA) or deoxyribose (found in
called guanosine. Nucleosides containing pyrmidines are
deoxyribonucleotides of DNA).
named with the sux "idine" at the end of the name of the
base they contain. Thus, the pyrimidine nucleosides are
4. The term nucleoside phosphate is equivalent to a

nucleotide (nucleoside + phosphate + base =
nucleotide). This is true whether it is a
monophosphate, diphosphate, or triphosphate.
5. The nitrogenous bases found in nucleotides
include adenine (purine), guanine (purine), thymine
(pyrimidine), cytosine (pyrimidine), and uracil
(pyrimidine).
6. The bases adenine, guanine, and cytosine are
found in both ribonucleotides and
deoxyribonucleotides. Thymine is almost always
found in deoxyribonucleotides. Uracil is found
The feudalist takes lots of notes
Everything from the baron he quotes
When it comes to election
The final selection
Depends on how evry Count votes
332
Round the household it was overheard

Causing sadness that no one preferred
Theres no guarantees
For this terminal disease
The calendars days are numbered
cytidine, uridine, and

thymidine.
8. Ribonucleotides
are the building
blocks of RNA and
deoxyribonucleotides are the building blocks of DNA.

9. Nucleotides and nucleosides are made in cells by two general
mechanisms - salvage pathways (use breakdown products of
other nucleotides/nucleosides) or de novo pathways
(synthesize nucleotides/nucleosides from scratch).
10. In salvage pathways, nucleic acids can be
broken down to nucleoside monophosphates
or individual bases. Monophosphates be
rephosphorylated to triphosphates by kinases
12. De novo synthesis of pyrimidines is fundamentally dierent

from that of purines. In purine biosynthesis, the base is
assembled on the sugar. In pyrimidine biosynthesis, the base is
made apart from the sugar and later attached to it.
13. In de novo synthesis of nucleotides, ribonucleotides are
synthesized first. Deoxyribonucleotides are made from
ribonucleoside diphosphates. Atoms in the ring of pyrimidines
come from aspartate and carbamoyl phosphate.
14. The enzyme carbamoyl phosphate synthetase has an
interesting catalytic strategy involving channeling of the
I confess that I truly cant see

Solving problems mathematically
So dont ask me then
How to double 2n
Cuz it all seems so 4n to me
in order to reincorporate them into nucleic

acids. Alternatively, nucleoside monophosphates can lose a
substrates through the enzyme as catalysis

occurs. This is important because some of the
intermediates (such as carboxyphosphate and
carbamic acid) are very unstable in aqueous
solution.
15. The most important regulatory enzyme for the entire pathway
phosphate (becoming nucleosides) or can lose the phosphate
of pyrimidine biosynthesis is aspartate transcarbamoylase
and the sugar to become a base. Bases can either be broken
(ATCase), which plays an important role in balancing the
down or reconverted back to nucleoside monophosphate by
amounts of purines and pyrimidines and also measuring the
addition of appropriate sugars and/or phosphates.
amount of energy available (via the amount of ATP present).
11. De novo synthesis of nucleotides utilizes very simply

precursors - amino acids, carbamoyl phosphate, and sugars.
Activated carbons are donated by folate derivatives.
The enzyme catalyzes

the linkage of aspartate
to carbamoyl phosphate
and is allosterically
activated by ATP and
At the dance I was wanting for sure

To step out with a lass I prefer
With my leg in a cast
I couldnt dance fast
But I sure had a crutch on her
333
diphosphate, but all diphosphates (purines, pyrimidines,

and all deoxyribonucleoside diphosphates) are converted
to triphosphates by NDPK.
18. Conversion of UTP to CTP is catalyzed by the
enzyme CTP synthase and it is inhibited by CTP, thus
providing a balance between the amount of CTP and
UTP.
19. De novo synthesis of purines uses atoms from
aspartate, glycine, glutamine, carbon dioxide, and
tetrahyrdofolate derivatives to make the purine ring.
20. The most important regulatory enzyme for the first
part of purine biosynthesis is PRPP amidotransferase. It
is inhibited fully by AMP and GMP, but is only partly
inhibited (is still partly active) when only one of these
molecules is present. This enzyme helps to control purine
production and also slows purine production when one
nucleotide gets to be in too high of a concentration.
allosterically inactivated by CTP.
21. In the process of making IMP, fumarate is released,
16. The first pyrimidine nucleotide made in the de novo pyrimidine

pathway is UMP.
17. UMP is phosphorylated to UDP (by uridine monophosphate
kinase) and then to UTP (by nucleoside diphosphokinase =
NDPK) before conversion to CTP. Each nucleoside
monophosphate has a specific kinase to convert it to the
thereby connecting purine biosynthesis to the citric acid cycle.

22. The first purine-like
intermediate in de
novo purine
biosynthesis is
inosinic acid (or IMP),
All American shoppers recall

Gazing out at the great urban sprawl
It really seems lame
Cuz they all look the same
See one center and youve seen a mall
334
which has the purine-like base hypoxanthine linked to ribose

(and ribose is linked to phosphate).
23. IMP can be converted to AMP or GMP. The pathway by which
IMP leads to GMP is inhibited by GMP and uses energy from
24. The pathway to GMP involves oxidation, whereas the pathway

that leads to AMP uses aspartic acid to donate an amine and
fumarate is again released.
25. Nucleoside monophosphates from de novo synthesis can be
ATP, whereas the pathway where IMP is converted to AMP is
converted by kinases to nucleoside diphosphates and
inhibited by AMP and uses energy from GTP. Thus, the critical
nucleoside triphosphates. These enzymes include AMP kinase
balance of these two nucleotides is maintained using this
(also called adenylate kinase) and GMP kinase (also called
scheme.
guanylate kinase), which convert nucleoside monophosphates

to nucleoside diphosphates, and nucleoside diphosphate
kinase (NDPK), which converts all nucleoside
diphosphates to nucleoside triphosphates.
26. Ribonucleotide reductase (RNR) catalyzes the
formation of deoxyribonucleotides from ribonucleotides.
The substrates are ribonucleoside diphosphates (ADP,
GDP, CDP, or UDP) and the products are
deoxyribonucleoside diphosphates (dADP, dGDP, dCDP,
or dUDP).
27. RNR has two pairs of two identical subunits - R1
(large subunit) and R2 (small subunit). R1 has two
allosteric binding sites and the active site of the enzyme.
R2 forms a tyrosine radical necessary for the reaction
mechanism of the enzyme.
28. Ribonucleotide reductase is allosterically regulated
via two binding sites - a specificity binding site (binds
dNTPs and controls which substrates the enzyme binds

335
and which deoxyribonucleotides are made) and an activity
whereas the ACTIVITY SITE is the allosteric binding site for ATP
binding site (controls whether or not enzyme is active - ATP
or dATP.
activates, dATP inactivates).

29. Specificity sites act in a generally complementary fashion.
30. Synthesis of dTTP by the de novo pathway takes a

convoluted pathway from dUDP to dUTP to dUMP. The latter
Binding of
reaction here is catalyzed
deoxypyrimidine
dUTPase.
triphosphates to the
31. The de novo pathway for
specificity site
thymidine synthesis
tends to inhibit
converts dUMP to dTMP,
binding and
using a tetrahydrofolate
reduction of
derivative and the enyzme
pyrimidine
thymidylate synthase. In the
diphosphates at the
process, dihydrofolate is
enzyme's active site
produced and must be
and stimulates
converted back to
binding and
tetrahyrdolate in order to
reduction of purine
keep nucleotide synthesis
diphosphates at the
occurring.
active site. Binding

of deoxypurine
32. The enzyme involved in
triphosphates tends
the conversion of
to inhibit reduction
dihydrofolate to
tetrahydrofolate,
of purine
diphosphates and stimulates reduction of pyrimidine
dihydrofolate reductase (DHFR), is a target of anticancer drugs
diphosphates. Don't confuse the active site with the activity
which inhibit the enzyme. An inhibitor of DHFR is methotrexate
site. The ACTIVE SITE is where the reaction is catalyzed,
or aminopterin.
336
33. ATCase is regulated allosterically by ATP (activates) and CTP
34. PRPP amidotransferase is an important regulatory enzyme for
(inactivates). It is the most important regulatory enzyme in de
purine
novo pyrimidine biosynthesis and it helps to balance the
biosynthesis. It is
relative amounts of purines and pyrimidines. Another important
inhibited by AMP,
GMP, and IMP. If
AMP is low and
If you long for the beer drinking spaces

A brewery can be an oasis
Is a lager the tops
Because of its hops?
Decide on a case by case basis
GMP is high (or

vice-versa), the enzyme is reduced in activity, but still can
function. This is important to help increase the amount of the
other one, thus helping to balance AMP and GMP.
35. Salvage of purine nucleotides is important metabolically perhaps more so than salvage of pyrimidines. The enzyme
HGPRT is involved in the direct salvage of guanine nucleotides
and indirectly involved in salvage of adenine nucleotides
through IMP and hypoxanthine.
36. Breakdown of purines results in production of xanthine.
Oxidation of xanthine yields uric acid. This compound serves
an excretory role in birds and dalmations (among other
organisms). Uric acid is not very water soluble and can
precipitate out in nerve cells, causing the painful condition
regulatory enzyme in the pathway is CTP synthase, which is
Gout often
inhibited by CTP. This enzyme helps balance the relative
strikes in the big
amounts of CTP and UTP.
toe.
known as gout.
Mr. Ritz stood before all the backers

Thanking them for the six million smackers
It is rare when folks say
They rejoice every day
That youre gonna go even more crackers
37. Uric acid acts

337
as an antioxidant and may have protective roles against

diseases, such as multiple sclerosis. The disease is
successfully treated with allopurinol, which acts as a suicide
inhibitor of the xanthine oxidase enzyme.
38. Severe combined immune deficiency arises from a deficiency
of adenosine deaminase. In immune cells of patients with this
disease, dATP accumulates, shutting o RNR and stopping cell
division.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
338
Key Points - Nitrogen Metabolism

1. Reduction of nitrogen to a form useful for organisms (called
nitrogen fixation) is a dicult and energetically costly process.
Nitrogen fixation is made possible by bacteria that have an
enzyme known as the
4. Amino acids are grouped into families corresponding to the

precursors they are made from. Students should know the
amino acids that are simple transamination products glutamate (made from alpha-ketoglutarate), glutamine (made
from glutamate), aspartate (made from oxaloacetate), alanine
(made from pyruvate),
nitrogenase complex.
asparagine (made from

aspartate).
2. In nitrogen fixation, N2
is reduced to
5. Other reactions
ammonium ion (NH4+).
involving amino acids
The process
involve transfer of single
theoretically requires
carbons. This often
six electrons and 12
involves derivatives of
ATPs to make two
folic acid (folates). Note
ammonia molecules
that folates can donate
(NH3), but hydrogen
single carbons AND
gas is always
accept single carbons.
produced, so two
additional electrons
6. Humans require folates
and 4 ATPs are also
in their diet and do not
needed (giving a total
synthesize them from
of 8 electrons and 16
ATPs) .
3. Once reduced to ammonium ion, nitrogen can readily be
incorporated into the amino acids glutamate, and glutamine.
scratch. Bacteria, on the

other hand, make them
from scratch. An intermediate they use in this regard is paraamino-benzoic acid (PABA). Folates exist in two main forms for
our purposes - dihydrofolates and tetrahydrofolates.
339
The haters of music agree

It is not something they wish to see
From the sound of their talk
Whether Mozart or Bach
They hate violins on TV
7.Anticancer drugs
ketone body formation ( acetoacetyl-CoA and acetyl-CoA =
and antibiotics
ketogenic), glucose metabolism (oxaloacetate or pyruvate =
sometimes target
glucogenic), or both.
folates. Bacterial cells

start synthesis of
folates with para-amino benzoic acid and drugs that mimic this
are the sulfa drugs. Humans get folate in their diet, so we are
not susceptible to action of these drugs. Methotrexate is an
anti-cancer drug that mimics folate.
as S-Adenosyl-Methionine (SAM). It serves as a

methyl group, it forms S-Adenylhomocysteine
(SAH) that can be readily broken down to
homocysteine. Elevated levels of homocysteine
Note how an amine group of aspartate is transferred to form

urea. Students should know the names of the molecules of the
pathway, as well as the pathway's involvement in making urea
by splitting it o of arginine. Note also that the cycle occurs
partly in the mitochondria and partly in the cytoplasm.
8. Another molecule involved in single carbon reactions is known

donor of methyl groups. After SAM donates its
11. The Urea Cycle is involved in nitrogen metabolism in cells.
12. Uric acid and urea are excretory forms of nitrogen. Human
Geriatrics are quick to address

A problem that causes distress
In turning the page
Of increasing ones age
Its not fun to feel so youthless
in the blood are associated with atherosclerosis.
primarily excrete urea, whereas birds (and

dalmations) excrete uric acid. Uric acid,
which is a breakdown product of guanine, is
NOT very soluble in water and can form
crystals, causing the disease known as gout.
Reduction of homocysteine in the blood is accomplished with

supplements of folic acid and, to a lesser extent, vitamins B6
and B12.
9. Essential amino acids for an organism are those amino acids
that the organism cannot synthesize themselves and must be
in their diet. Humans have 10 amino acids considered
essential.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
10. Breakdown of amino acids (catabolism) is divided into those

amino acids whose carbon backbone forms intermediates in
340
DNA Replication, Repair, Recombination

1. DNA consists of a double helix. Each strand of the helix is a
polymer of nucleotides joined together in phosphodiester
linkages that have
3. DNA contains four bases - A,T,C, and G arranged with A paired

with T and G paired with C on the internal portion of the double
helix. Hydrogen bonds stabilize these base pairs - two for the
A-T pair and three for the G-C pair. Thus, G-C pairs are harder
to break than A-T pairs.
alternating sugarphosphate-sugar-
4. DNA has a major and
phosphate links. On the
a minor groove arising
inside of the double helix
from asymmetric
are the complementary
glycosidic linkages
base pairs held together by
between the
hydrogen bonds.
deoxyribose sugar and

each base in the double
2. The arrangement of the
helix.
double helix is in an 'antiparallel' fashion, meaning
5. DNA has three major
that one strand oriented in
forms - A,B, and Z. The
the 5' to 3' direction is
A and B forms are right-
directly paired to a
handed helices, whereas
complementary strand
the Z form is a left-
oriented in the 3' to 5'
handed helix. The B
direction. Phosphodiester
form of DNA is the most
bonds involve linkage
prevalent one and
contains about 10.5
between the 5' phosphate

group of the incoming nucleotide and the 3' hydroxyl of the
previous nucleotide in the chain.
bases per turn of the helix.

6. Z-DNA may have roles in marking the location of genes in
eukaryotic chromosomes.
341
7. Another DNA form is the A form (actually discovered by
stranded region for replication. The 5' to 3' syntheses on the
Rosalind Franklin), which is more "compressed" and is also a
lagging strand are discontinuous. The many pieces of lagging
right-handed helix. The A form is the form assumed by double
strand synthesis are called Okazaki fragments.
strand RNA or RNA-DNA duplexes as well. RNA cannot exist in

the B form due to steric hindrance arising from the oxygen on
carbon number 2 of ribose, which is not
11. Okazaki fragments must be combined together ultimately.

First, the RNA primer must be removed from each one. The 5'
present in the deoxyribose of DNA.

8. All DNA polymerases require a primer
to start DNA synthesis. The primer is
formed inside of cells by a special RNA
polymerase known as primase. (RNA
polymerase does not require a primer)
9. DNA replication proceeds by two
distinct mechanisms (both 5'-3',
however)- one on each strand. Leading
strand and lagging strand synthesis
occur by dierent mechanisms, but
both are catalyzed by the same DNA
replication complex (Pol III, in the case
of E. coli).
10. Leading strand synthesis is
continuous in the 5' to 3' direction.
Lagging strand synthesis can only
occur when the leading strand
synthesis opens up a new single-
342
The work of the dentist in serving

His patients was always unswerving
He got mighty praise
Working with the decays
But the root canals all were unnerving
to 3' exonuclease
15. DNA Polymerase III is very processive in its action, meaning
activity of DNA
that once it gets onto a DNA molecule, it stays on it for a long
Polymerase I is
time replicating it. DNA Polymerase I is NOT very processive.
needed to remove the
The dierence between the processivity properties of these two
initial RNA primer of
enzymes is because a protein called a sliding clamp (beta
leading strand synthesis, but is needed frequently to remove
clamp in E. coli) holds DNA polymerase III to the DNA during
the primers of lagging strand synthesis.
replication, but it does not interact with DNA Polymerase I.
12. DNA ligase is an enzyme that creates phosphodiester bonds
16. In E. coli DNA replication, a dimer of DNA Polymerase III is at
between adjacent nucleotides between Okazaki fragments.
the replication fork and performs most of the DNA replication in
Biotechnologists use this enzyme to join DNA fragments
the cell. One portion of it replicates the leading strand and the
together to create recombinant

molecules.
13. E. coli DNA replication occurs at 1000
base pairs per second. At 10 base pairs
per turn, this represents a machine
other replicates the lagging strand. Leading

When the piracy expert exploded
All the support that he had was eroded
The cause of the storm?
He tried to stay warm
By wearing a jacket down-loaded
turning at 5000 to 6000 rpm. E. coli's
strand synthesis is faster, so the lagging strand

template sometimes loops out in a trombone-like
fashion when the lagging strand replication falls
behind.
17. Proteins at/near the replication fork and their
helicase protein (DNA B - part of the BC complex) unwinds
functions include primase (makes RNA primers necessary for
DNA at a rate of at least 5000 - 6000 rpm. The protein
the DNA polymerase to act on), SSB (single stranded binding
separates strands ahead of the DNA Pol III so as to make
protein - protects single-stranded DNA and interacts with the
single strands accessible for replication.
replication proteins), DNA gyrase (topoisomerase II - relieves
14. Unwinding of strands causes superhelical tension to increase

ahead of the helicase. Topoisomerase II (gyrase) relieves the
tension created by the helicase and is essential for replication
to proceed eciently.
the superhelical tension created by helicase), Pol I (removes

RNA primers),
DNA ligase
(joins DNA
fragments
together by
At Georgia Pacific the labor

Was performing an odd kind of caper
For the tiny and svelte
They made tissue belts
Out of something they dubbed the waist paper
343
catalyzing synthesis of phosphodiester bonds at nick

sites), and helicase (unwinds double helix).
18. DNA Polymerase I is much more abundant in cells
than DNA Polymerase III and it functions to remove
RNA primers on Okazaki fragments and fill in the short
regions where the RNA bases were with DNA.
19. DNA polymerase I has three enzymatic activities - a
5' to 3' DNA polymerase activity, a 3' to 5' exonuclease
activity (also called proofreading), and a 5' to 3'
exonuclease activity.
20. All DNA polymerases require a primer to start DNA
synthesis. The primer is formed inside of cells by a
special RNA polymerase known as primase. (All RNA
polymerases do not require a primer)
21. Removal of RNA primers in E. coli requires DNA
PolymeraseI's 5' to 3' activity. Without it, the cell will
die.
Writhes can be positive or negative and in either case, when
22. The linking number (L) of a DNA is the sum of the number of
twists (T) of a DNA plus the number of writhes (W). Thus, L = T
+ W. The twists are the number of times two the two helices
The vegetarian editor takes
Very long evening mealtime breaks
And wears a big frown
Every time she sits down
Perhaps its because of missed steaks
cross each other. The

writhe is the number
of superhelical turns
found in a DNA.
the W is a non-zero value, the molecule is said to be

superhelical or to have superhelicity.
23. Writhing of DNA occurs in an attempt by a DNA molecule to
"relax." A DNA molecule is relaxed when its number of base
pairs (bp) per twist (T) is that of B-DNA (10.4-10.5 bp per turn).
Thus, if one takes a relaxed circular DNA, opens it and removes
two twists from it and then closes it, the number of twists will
344
decrease, but the number of base pairs remains the same. In

this case, the numbers of bp per twist will INCREASE. This
causes a tension that is relieved by the
DNA TWISTING two turns. This will
cause the writhe to compensate by
forming two negative superhelical turns,
giving W a value of negative two. Note
single-stranded regions and cause DNA A

protein to be released. The primases begin
The primases DO NOT require a pre-existing primer to function.
it and adds twists to it and then closes it, the number of twists
will increase, but the number of base pairs remains the same.
In this case, the numbers of bp per twist will DECREASE. The
25. Initiation of replication in E. coli occurs
28. Next, SSB and primase bind the exposed
RNA synthesis only also) in opposite directions on each strand.
24. On the other hand if one takes a relaxed circular DNA, opens
increase to a value of positive two.
protein is released in the process.
synthesizing RNA primers (remember - 5' to 3'
same.
turns, which will cause the writhe
each of the single strands in opposite orientations. The dnaC
Old Donald the Disneyland gent

Hurt his foot when it banged the cement
Still he danced with much pain
And he never complained
To set a lame duck precedent
that the linking number remains the
DNA will relax by UNTWISTING two
27. Next, the dnaBC complex binds the dnaB protein (helicase) to
29. Note that replication of the E. coli circular DNA is bidirectional - two replication forks pointed in opposite directions
from the origin. They meet later at a termination site on the
other side of the genomic DNA.
The cereal farmer did cry

Of genetical problems heres why
His crop it appeared
Was re-engineered
So all of his plants turned a-rye
at a specific site on the E. coli genomic

DNA, known as OriC, in the cell's circular chromosome. The
OriC site contains three repeats of an AT rich sequence near
some sequences bound by the DNA A protein.
26. Replication initiation begins with binding of the several copies
of the dnaA protein to the OriC site. Bending and wrapping of
the DNA around dnaA proteins causes the AT-rich sequences
30. Eukaryotic DNA replication is coordinated

tightly with the cell cycle. Checkpoints during the
cell cycle ensure that progression through the cell
cycle does not occur if there are problems with
the DNA.
31. When such conditions arise, the repair process can be

initiated and if repair cannot be performed, a series of events
resulting in cellular death may start to occur.
32. Eukaryotic chromosomes dier from prokaryotic DNAs in
being linear. The linear ends of the chromosomes are called
noted above to become single-stranded.

345
telomeres. Telomeric sequences have thousands of copies of

repeats of short sequences.
33. The enzyme that builds telomeres is called telomerase and is
found predominantly in fetal and cancer cells, as well as
fertilized eggs.
36. Damage to DNA can occur chemically (deamination of

adenine to form hypoxanthine), by oxidation (creation of 8-oxoguanine by reactive oxygen species reaction), by reaction with
an aflatoxin metabolite, by reaction with a cross-linking
reagent, such as psoralen, and by dimerization of adjacent
thymines stimulated by
Dierentiated cells for
ultraviolet light.
the most part do not

appear to have an
37. These systems
active telomerase.
require repair - described

below. Another system
34. With each round of
requiring repair is DNA
DNA replication, linear
sliding, which can occur
chromosomes in
amid repeating
eukaryotes shorten.
sequences. Lack of a
Thus, the longer the
repair system for these
telomeric sequences
leads to Huntington's
are in a chromosome,
disease.
the more divisions it

can undergo before
38. DNA Repair systems
the telomeres are
to repair damage include
"eaten up".
35. Telomerase is a
A. Proofreading
(technically only repairs
reverse transcriptase -
mismatches, not
an enzyme that uses an RNA template (a circular RNA that it
damage) was discussed earlier. It involves 3'-5'
carries) to synthesize DNA. Other reverse transcriptases are
exonucleolytic activity of a DNA polymerase and it occurs as
found in retroviruses, such as HIV.
DNA is being replicated.

346
B. Mismatch repair - fixing of
40. Nucleotide excision repair can be
mismatches that occur, largely
used to repair thymine dimers. Here, a
as a result of errors in
segment of DNA containing the damage
replication.
is removed by uvrABC excinuclease. The

gap is then filled in using DNA
C. Nucleotide Excision Repair
Polymerase I and DNA ligase.
- Excision of a group of
nucleotides followed by
41. Base excision repair can removed
replacement with correct ones
damaged based from DNA. It diers from
by DNA polymerase. Removal
nucleotide excision repair in removing the
of thymine dimers, for
damaged base first, followed by removal
example
of a segment where the base was.
D. Base excision repair -
42. Disruption of error correction systems
Excision of a damaged base -
can have severe consequences.
for example uracil glycosylase
43. Error-related systems associated with
removal of U from DNA.
cancer include HNPCC (colon cancer)
39. In E. coli, mismatch repair
and BRC-A, which is involved in DNA
occurs as a result of action of the
repair. A critical protein for monitoring
proteins MutS (recognizes
DNA for damage prior to division is p53.
mismatch), MutL (recruits MutH),
It can stop the cell cycle if it senses
and MutH (nicks newly
damage and initiate repair. If repair is
synthesized strand of DNA to
unable to be performed, p53 can induce
allow exonucleolytic removal of
cellular suicide - apoptosis.
nucleotides around the

mismatch).
44. An Ames test uses a selectable

marker that can give a readily observable
phenotype (such as growth on antibiotic) when mutation
347
46. Recombination proceeds through formation of a

Holliday junction. Holliday junctions form as a result of
alignment of homologous sequences, followed by
cleavage of strands on each chromosome, invasion of the
strands into the opposite chromosome, movement of the
junction, another cleavage reaction, followed by
reformation of phosphodiester bonds.
47. Enzymes involved in recombination are called
recombinases and are similar in function to the integrase
of HIV.
Im wondering what I should do
For my dentist whos feeling quite blue
Her work with decays
Has caused a malaise
What is more, she has got fillings too
happens. By comparing the number of cells with the

observable phenotype in a the presence of a test compound to
the number of cells in another tube lacking that compound, the
mutagenicity of a compound can be determined.
45. Recombination of DNA results in mixing and matching of DNA
sequences. The process occurs most often between
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
homologous sequences on dierent chromosomes. The

process can be quite active during meiosis.
348
Key Points - Transcription

1. Transcription is the process where RNA is made using DNA as
a template. Students should ABSOLUTELY not mix up or
misuse the terms DNA Replication, Transcription, and
Translation.
2. RNA polymerization requires an enzyme called RNA
polymerase. It can start a chain without a primer, incorporates
5. In E. coli, all of the RNAs are made by a single polymerase,

known as RNA Polymerase. Eukaryotic cells have three RNA
polymerases - RNA Polymerase I (rRNAs), RNA Polymerase II
(mRNAs and snRNAs),
Perhaps it was heavenly love
Or coincidences thereof
That gave Matt a B
In trigonometry
He thinks twas a sine from above
nucleotides into a growing chain in the 5' to 3' direction using

phosphodiester bonds, and uses ATP, GTP, CTP, and UTP as
starting compounds. The product of RNA polymerization is
called a transcript.
3. The 5' -most nucleotide in RNA has three phosphates on it. All
other nucleotides in RNA have only the single phosphate of a
phosphodiester
bond. Synthesis of
the phosphodiester
bond arises from
nucleophilic attack of
Perhaps he was being perverse

Using mirrors while writing so terse
Wouldnt you know it
This most backwards poet
Did all of his writing inverse
and RNA Polymerase

III (tRNAs).
6. E. coli RNA
Polymerase has five
distinct polypeptide
subunits we discussed in class - alpha, beta, beta prime, and

sigma.
7. Footprinting is a technique for determining where on a DNA
molecule a protein is bound.
8. Promoters in E. coli (RNA polymerase binding sites adjacent to
genes) function with widely varying rates of eciency - some
stimulating initiation of transcription every few seconds, others
only one or twice per life cycle. One way for a promoter to
control such events is via variation in conserved sequences. E.
the 3' oxygen on the internal phosphate (closest to carbon 5 of
coli genes have two conserved sequences - one at -10 relative
the ribose) of the incoming 5' nucleotide.
to the transcription start site (TATAAT) and another at -35
4. Cells have three main types of RNA - mRNA (carries message

to be translated into protein), tRNA (carries amino acids to
relative to the transcription start site (TTGACA).

9. The more closely a given promoter's sequence matches the
ribosomes for incorporation into protein), and rRNA
consensus sequence of the -10 sequence, the more active the
(components of ribosomes). Small RNAs are a fourth type of
promoter is at initiating transcription. RNA Polymerase slides
RNA that play important roles in control gene expression.

349
along the DNA and when the sigma subunit identifies a

promoter site, it stops.
13. The strand of DNA that is NOT copied in transcription is

called the coding strand, since it has the same sequence as the
RNA except for having T instead of U.
10. Dierent sigma factors (such as

the one made during heat shock)
14. Transcription occurs in three
allow the cell to turn on sets of
phases - initiation, elongation, and
genes with dierent sequences in
termination. The initiation phase
the -10/-35 sequences as needs
creates a open transcript complex and
arise.
this phase can be inhibited in

prokaryotes by rifampicin. During the
11. Transcription occurs in a DNA
initiation phase, the transcription
"bubble" in which the RNA is partly
bubble is formed and the first ten or so
base paired to the template strand,
nucleotides are polymerized together
with the 5' end of the RNA
by RNA polymerase. During the
"hanging" o. The bubble moves as
initiation phase, the sigma factor is
the RNA polymerase moves. Ahead
released from RNA polymerase.
of the bubble, positive supercoils

are created. This superhelical
15. Transcription occurs under the
tension is relieved by DNA Gyrase.
'control' of promoters. The more
As the strands come back together
strongly RNA Polymerase (in
behind the bubble, negative
prokaryotes) binds to the promoter, the
supercoiling occurs, and this is
more transcripts are made of that
relieved by a topoisomerase.
particular gene. The more transcripts

are made, the promoter is referred to
12. The strand of DNA being copied is
as being 'stronger.'
known as the template strand. It

runs 3' to 5', since the RNA is being
made 5' to 3' complementary to it.
16. Transcription termination occurs
by both factor dependent means

350
Henry Ford wins the prize, but of course

For breaking a rule some endorse
His particular zeal
For the automobile
Made him put his cars fore the horse
(requires a protein
message is being transcribed. There are no significant
factor) and factor
modifications to mRNAs in prokaryotes.
independent
means (no extra
protein required).
The factor independent method relies on formation of a duplex

sequence of GC base pairs immediately ahead of a stretch of
U's. The duplex destabilizes the RNA-DNA duplex and this is
favored by the relatively weak hydrogen bonds of the U-A
interactions.
17. The factor involved in factor dependent transcription
termination in E. coli is called rho. It binds to the 5' end of an
RNA being made and (using ATP energy) "climbs" the RNA until
it reaches the RNA polymerase. There it destabilizes the RNA/
DNA duplex, favoring the release of the RNA polymerase from
the DNA and the RNA from the DNA, as well.
18. In prokaryotes, tRNAs are the most altered (processed) RNAs.
Modifications start with their being cleaved from a larger RNA
containing both tRNAs and rRNAs. Ribonuclease P is a
ribozyme (catalytic RNA) that cleaves the 5' end of tRNAs from
the larger RNA. Ribonuclease III catalyzes excision of rRNAs
from the larger molecule.
19. Eukaryotes and prokaryotes dier significantly in the
relationship between transcription and translation. Prokaryotes
have no nucleus. In them, translation starts oftentimes WHILE a
351
20. In eukaryotes, transcription and translation are spacially

separated. Transcription occurs in the nucleus, whereas
translation occurs in the cytoplasm. In addition, eukaryotic
mRNAs are modified at the 5' end (capping), the 3' end
(polyadenylation) and even in the middle (editing and splicing).
21. Eukaryotes have 3 specialized RNA polymerases. They dier
in their sensitivity to alpha-amanitin (a poison from some
mushrooms). RNA polymerase II (makes mRNAs) is the most
sensitive. RNA polymerase III (makes tRNAs and small rRNA)
has moderate sensitivity and RNA polymerase I (makes large
rRNAs) has low sensitivity.
22. Sequence elements aect transcription of eukaryotic genes.
They include the TATA box (positioned approximately -30 to
-100), and a CAAT box and GC box (-40 to -150).
23. The TATA box is not found in front of all eukaryotic genes, but
is essential for strong transcription.
genes located up to many thousands of base pairs upstream
24. The promoters for each RNA polymerase are dierent in
(ahead of), downstream (down from ) or even in the middle of

genes.
structure.
25. Enhancer sequence elements are DNA sequences that are
The geology students bemoan
Each exam has the same overtone
The grading is picky
And the questions are tricky
As if they were written in stone
26. RNA Polymerase II in eukaryotes diers from RNA polymerase
bound by enhancer
in E. coli in not binding to the DNA directly, but rather, it must
(transcription factor)
bind to another protein that binds to the promoter first.
proteins. Enhancer
27. Transcription factors assist RNA Polymerase II in binding. For
proteins act in this way to
genes with a TATA box, the transcription factor TFIID binds first.
enhance transcription of
It contains a subunit called TBP (TATA-Binding Protein) that

352
recognizes
When mom told him what would displease her

Little Brutus thought her an old geezer
When a chance would arise
He would spurn her advice
And so started running with Caesars
31. Splicing is a modification to eukaryotic RNAs that occurs in
and binds to
the middle of the RNA. Splicing occurs to mRNAs, tRNAs and
the TATA
rRNAs in eukaryotes.
sequence.
28. In
32. Splicing involves removal of internal sequences from RNA

followed by joining of ends. The removed sequences are called
eukaryotes, rRNA is made by the action of RNA Polymerase I. It
introns. The segments that make it into the final RNA are called
is made as a precursor and then processed, not unlike what
exons.
occurs in prokaryotes.
33. The only sequences common to all spliced RNAs are a GU
29. tRNAs in eukaryotes are made by action of RNA Polymerase

III. Processing of them includes removal of a 5' leader
sequence and part of a 3' tail, as well as
addition of a CCA sequence at the new 3'
end. The amino acid gets attached to the 3'
end of the tRNA.
sequence at the 5' end of the intron and an AG at the 3' end of
the intron. A third sequence - an A residue surrounded by
My wife said last night I did keep

Telling stories when counting the sheep
I said many things
From Lord of the Rings
Cuz I was Tolkien in my sleep
30.In contrast to prokaryotic mRNAs,

eukaryotic mRNAs are extensively modified. Modifications
include
pyrimidines also is common.

34. Protein/RNA complexes called
snRNPs mediate the splicing process in
higher eukaryotes. snRNPs contain small
nuclear RNAs (snRNAs) and proteins.
35. In splicing, the hydroxyl of the A residue attacks the

phosphate of the phosphodiester bond at the 5' end of the
a. Addition of a 5'-5' cap of a methyl guanosine to protect

from degradation
intron, creating a 5'-2' bond (part of the lariat structure). Attack
b. Addition of a poly-A tail at the 3' end under the control of

the sequence AAUAAA near the end of the mRNA (also to
protect from degradation)
joins the two exon ends and releases the intron as a lariat.
by the released 3' end of the exon on the 3' end of the intron
36. Exon
c. Editing - modification of bases chemically, such as was

described in class for the Apo-B proteins
shuing
d. Splicing - removal of introns between exons.
splicing in
(occurs in
Everywhere now it seems its the rage

Spending money as if to upstage
The neighbors next door
Who spend even more
These people should act more their wage
353
dierent tissues) allows cells to make many versions of protein

from a single sequence. This is important in immunology and in
fine-tuning cellular needs that are tissue specific.
37. Lower eukaryotes are able to excise introns by an
autocatalytic mechanism. At least one prokaryotic gene is
spliced autocatalytically.
38. In splicing, the U1 snRNA forms base pairing with the 5' end
of the intron sequence.
39. In splicing, the U2 snRNA froms base pairs with the
pyrimidine-rich region in the intron and with the snRNA of U6.
Pairing with the intron forces outwards the 'A' residue that
attacks the phosphate, as noted in class.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
354
Key Points - Translation

1. Translation (protein synthesis) is performed by
ribosomes on mRNA and occurs in the 5' to 3'
direction along the mRNA.
2. The rate of translation in bacteria is about the same as
the rate of transcription (45-50 bases or 15-17 amino
acids per second). The 5' end of the coding region
corresponds to the amino end of the protein. The 3'
terminus of the coding region corresponds to the
carboxyl end of the protein.
3. Translation is performed by ribosomes on mRNA and
occurs in the 5' to 3' direction. The rate of translation
in bacteria is about the same as the rate of
transcription (45-60 bases or 15-20 amino acids per
second).
4. The 5' end of the coding region corresponds to the
amino end of the protein. The 3' terminus of the

coding region corresponds to the carboxyl end of the protein.
5. As polypeptides are being synthesized, the previously
The teenagers friends were all crowing
Of the show to which they were a-going
Jan worried that she
Would be absentee
Because she had parents all no-ing
synthesized chain
is attached to the
free amine of the
incoming (new)
amino acid and the
entire complex is, as a result, attached to the 'new' tRNA.

Thus, polypeptides are synthesized in the amino to carboxyl
terminus.
6. Transcription and translation are coupled together in bacteria,
but not in eukaryotes.
7. Translational accuracy is about one error per thousand to ten
thousand amino acids. Greater accuracy would slow translation
355
down, so a balance is struck between the need for accuracy
tRNA) is called
and the need to synthesize proteins reasonably rapidly.
the wobble base
8. tRNAs have extensive, but short self-complementary regions.

tRNAs themselves are typically 73-93 bases long, with duplex
regions at least partly in the A form.
The heir didnt think it was great

With his uncle now at heavens gate
He got zero stocks
Just a hundred old clocks
So hes now winding up his estate
because it is
less important
for specifying
the amino acid to be inserted than the first two bases.
9. The shape of tRNAs is that of an 'L'. At the 5' end of the tRNA
is usually a 'G' and at the 3' end is a CCA.
13. Aminoacyl-tRNA synthetases have the ability to recognize and

correct errors in joining of amino acids to tRNAs. For example,
10. Enzymes that catalyze the linkage of amino acids to tRNA's 3'
if one puts the wrong amino acid on the end of a tRNA and
or 2' ends are called aminoacyl-tRNA-synthetases. There are
then adds an appropriate aminoacyl-tRNA synthetase, the
20 of these enzymes - one for each amino

acid.
11. Amino acids are linked to tRNAs by ester
bonds between the carboxyl group of the
amino acid and either the 2' or 3' hydroxyl
The hypnotist took up his station

With a dentist to ease the sensation
The number one question
Will the power of suggestion
Let a trance end dental medication?
of the ribose of the terminal adenosine residue of the tRNA.

12. The anticodon loop has three bases complementary to the
codon in the mRNA. tRNAs provide the translation function
between nucleic acid sequence and amino acids. The
anticodon loop frequently contains the inosine base. The base
An ornithological gent
Had his day truly filled with lament
To make a new sweater
He required some goose feathers
But there was a large down payMENT
at the 3' end of the

codon of the mRNA
(corresponds to the
amino acid is readily removed.

14. Two regions of aminoacyl-tRNA
synthetases are important for editing - called
the activation site and the editing site.
15. There are two classes of amino acid
tRNA synthetases. They dier in the way they bind tRNAs and
in which hydroxyl of the ribose ring they attach the amino acid
to.
16. Class I amino acid tRNA synthetases attach the amino acid to
the hydroxyl on carbon #2. Class II enzymes attach the amino
acid to the hydroxyl on carbon #3.
17. Base pairings in RNA are slightly dierent than in DNA. For
base at the 5' end of
example, G-U base pairs are not unstable. "I" (inosine) can also
the anticodon in the
pair with C,U, or A.

356
18. In the genetic code, there are 64 possible combinations of the
21. Formylation of methionine in prokaryotes protects the
bases of the codon. Three of the possibilities (UAA, UGA, and
otherwise free amino end from reacting intramolecularly and
UAG) are used as 'stop' codons. They tell the ribosomes where
terminating transcription.
to stop making protein. A start codon is AUG and it codes for

methionine. Since there are 61 codons used to code for amino
acids and there are 20
22. Peptides exit the ribosome as they are being synthesized via
a tunnel in the structure.
amino acids, there is
23. Ribosomes have
therefore 'redundancy'
three sites for binding/
in the genetic code.
holding/releasing tRNAs.
They are called the A,P,
19. The Shine-Dalgarno
and E sites,
sequence (GGAGG) is
corresponding to the
located near the AUG
order in which tRNAs
start codon in
move through them
prokaryotic
(except for the very first
sequences. It is
one known as the
complementary to a
initiator tRNA = Met-
sequence in the 16S
tRNAf).
rRNA and serves to

help align the
24. Initiation of protein
ribosome with the start
synthesis starts with
site for translation in
binding of IF1 and IF3 to
prokaryotes.
20. In prokaryotes, the first amino acid incorporated into a protein
the 30S ribosomal unit.

25. IF2 (when bound to
is a formylated form of methionine called fMet. The formyl
GTP) acts to carry the Met-tRNAf to the P site of the 30S
group is put onto methionine after it is in the tRNA by a
subunit and base pairs it with the AUG start codon. IF3 departs
transformylase enzyme.
357
27. The process of elongation begins on the 70S

initiation complex. EF-Tu (a G protein coupled to GTP)
carries a charged tRNA to the A site of the complex. If
the tRNA anti-codon base pairs properly with the codon
in the mRNA, it stays matched with the codon and GTP
is hydrolyzed on EF-Tu and EF-TuGDP is released. If the
tRNA anti-codon does not form a stable base pairing
with the complex, the entire charged tRNA-EF-Tu-GTP
complex dissociates.
28. Next, the peptide group on the tRNA in the P site is
transferred and covalently linked via peptide bond to the
amino acid on the tRNA in the A site. This reaction is
catalyzed by an enzymatic activity called
peptidyltransferase - a ribozyme activity of the 23S rRNA
in the 50S subunit.
29. The tRNA in the A site along with the peptide it is
in the process. The complex of mRNA, IF1, IF2, and Met-tRNAf

is called the 30S initiation complex.
26. Hydrolysis of the GTP in IF2 results in release of the IF2 and IF1
from the initiation complex. That, coupled with binding of the
50S subunit yields the 70S initiation complex with Met-tRNAf in
the P site and the A and E sites open.
covalently attached to is transferred to the P site as the

"empty" tRNA in the P site is moved to the E site. EF-GGTP is involved in the process and GTP is hydrolyzed in the
process. EF-G-GTP has a similarity to the tRNAaminoacid-EFTu-GTP complex and may act to displace it.
30. As the old
tRNA is
released from
the E site, the
The Dominoes patrons perchance

Had good reason for looking askance
When a big no-no
Came from Yoko Ono
Who asked them to give pizza chants
358
At the meteorologists fair

Each attendee was made most aware
Though they always want surety
Theres no job security
Since their future is up in the air
empty A site accepts
the membrane, or outside of the cell, starts during protein
the aminoacyl tRNA
synthesis. Proteins destined to leave the cytoplasm have a
corresponding to the
signal sequence, consisting of a stretch of hydrophobic amino
next codon. The net
acids near their amino terminus.
result of one turn of
this cycle is that the polypeptide has grown by one amino acid
residue and the ribosome has moved along the mRNA by three
nucleotide residues. The process is repeated until a termination
signal is reached.
35. When the signal sequence emerges from the ribosome during
translation, it is recognized by the signal recognition particle
(SRP), which takes the entire ribosome/mRNA/polypeptide
complex to the endoplasmic reticulum. There it interacts with
the SRP receptor and in the process it links the ribosome with
31. The process of translation termination begins when a stop
the emerging polypeptide sequence to the translocon.
codon appears in the A site of the ribosome.

Termination of translation requires action of
release factors (RF1, RF2).
32. RF1 and RF2 carry water to the A site. The
peptidyl transferase transfers the
The farmer just lost a big frown

Because his lost cow has been found
When asked to define
How he got his bovine
He said that he just tractor down
polypeptide on the tRNA in the P site to

water, thus releasing the completed polypeptide from the
ribosome.
33. Eukaryotic translation is mechanistically similar to prokaryotic
36. The polypeptide passes through the

translocon channel as it is being made and
when the signal sequence completely exits the
translocon, a signal peptidase clips it free of
the rest of the polypeptide. The translation
process continues until the stop codon is
reached and then everything releases from the translocon.

37. The polypeptide remains in the endoplasmic reticulum where
it is further process to travel to the Golgi for additional
translation. Dierences include ribosomes (40S vs 30S and 60S
processing and targeting. Other sequences in the polypeptide
vs 50S), rRNAs (28S, 18S, 5.8S, and 5S), mRNAs (cap at 5' end
may help to
and polyA at 3' end, both involved in translation), and lack of
direct
formyl group on initiator tRNA's methionine.
modifications
34. Other translational dierences in eukaryotes relate to the

structure of eukaryotic cells. Targeting of proteins to organelles,
and/or the final

destination of
For a child acting out its transparent

Theyre rewarded for acting aberrant
If you question the cause
Of the childs many flaws
The answers most certainly apparent
359
the mature protein.

38. Antibiotics frequently are designed to target various aspects
of translation. They include streptomycin (interferes with
binding of formylmethionyl tRNA to ribosome), tetracytline
(inhibits binding of aminoacy-tRNAs), chloramphenical (inhibits
peptidyl transferase activity), and puromycin (causes premature
chain termination).
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
360
in adjacent alpha helices and
Key Points - Gene

Expression
contain regions with leucine

residues appearing about every 7
1. Gene expression refers to the
amino acids. The leucine interact
processes that result in the
with each other to hold the
production of functional protein.
strands together and in doing so
Gene expression can be controlled
allow other portions of the helix to
at the levels of transcription,
bind DNA properly.
processing (splicing in eukaryotes),

translation, mRNA stability, and
4.Zinc fingers are structures with
protein stability. Tissue-specific
cysteine residues that hold zinc
gene expression is essential for
ions and create a finger-like
multicellular, dierentiated
structure that can stick into the
organisms.
DNA helix.
5. Proteins that bind to specific
2. Transcription factors, as noted

previously, are proteins that bind to
DNA sequences must "read" the
DNA and aect the transcription of
sequence of bases inside the
genes located near where they
helix, usually by inserting a
bind. Common DNA-binding
portion of themselves into a
structures are found in the diverse
groove of the DNA and assessing
set of transcription factors that are
the hydrogen bonding molecules
know. They include motifs (motifs -
inside. Since dierent base pairs
structural features) for helix-turn-
have unique hydrogen bonding
helix, homeodomains, leucine
orientations, the proteins find and
zippers, and zinc fingers.
bind to specific base sequences.
3. Leucine zipper structures are found
6. Control of gene expression is

361
I just got mad and I cursed

When I learned that my lunch plans reversed
Someone buried my brat
Underground in a spot
And so I exhumed all the wurst
also essential for
when acted on by beta-galactosidase, giving a measure of how
prokaryotic
much the operon has been induced by the amount of blue
organisms to be
color produced.
able to respond
properly to their
environments. For example, E. coli prefers glucose for energy,

but must be able to use other sugars, like lactose, when they
are available.
7. An operon is a prokaryotic system for organizing genes all
under the same transcriptional control. Genes on the same
operon in prokaryotes are all synthesized on the same mRNA.
mRNAs containing multiple gene coding sequences are
referred to as polycistronic.
8. The lactose operon consists of three linked structural genes
that encode enzymes of lactose utilization, plus adjacent
regulatory sites. The three enzymes --z, y, and a--encode betagalactosidase, beta-galactoside permease (a transport protein),
and thiogalactoside transacetylase (an enzyme of still unclear
metabolic function), respectively.
9. Transcription of the lac operon commences at a promoter
(lacP) before lacZ and transcribes a 5,200 nucleotide
messenger RNA molecule (mRNA), ending at a terminator
beyond lacA.
10. X-Gal is a synthetic substance used to study lac operon
expression. X-Gal has the useful property that it turns blue
11. Negative transcriptional regulation of the lac operon is

accomplished by a protein known as the lac repressor. It binds
the operon's operator region and inhibits transcription.
12. In the absence of inducer molecules, the lac repressor tightly
binds to the operator and inhibits transcription of the operon.
When inducer molecules are present, they bind to the lac
repressor and change its shape and reduce its ability to bind
the operator, thus allowing the RNA polymerase to bind the
promoter and start transcription.
13. The promoter sequence of the lac operon diers somewhat
from the ideal consensus sequence of an E. coli promoter.
Consequently, in the absence of positive acting elements, the
lac promoter does not function well on its own. A protein that
acts positively to help activate the lac operon is the CRP
(cAMP Receptor Protein).
14. CRP (also called CAP) must bind to cAMP in order to
function. When CRP binds cAMP, its anity increases for the
lac operon adjacent
to the RNA
polymerase binding
site (-68 to -55). This
binding facilitates
Master gardeners think it is wise

So listen when these folks advise
Theres no number one
With just a green thumb
You also need two fertile eyes
362
transcription of the lac operon by stimulating the binding of

RNA polymerase to begin transcription.
15. When both CAP and the lac repressor are bound to the lac
operon, the repressor 'wins', shutting down transcription of the
19. Histones of the octamer have strong structural similarity to

each other.
20. Wrapping of DNA around the histone octamer provides only
partial compression of the length of a DNA molecule. Additional
operon.
compression occurs as a
result of coiling of octamer/
16. In eukaryotic cells, DNA
DNA complexes as well,
is wrapped up (coiled)
forming higher order
with basic proteins called
structures.
histones. Histone
sequences are strongly
21. Enhancer sequences
conserved from yeast to
(also called sequence
humans.
elements) are bound by

enhancer proteins and are
17. Four histones form a
found only in eukaryotes.
core around which DNA
Multiple enhance
is wrapped. This core
sequences may be present
contains two copies
before the start site of a
each of histones H2A,
particular gene. Binding of
H2B, H3, and H4. This
enhancer proteins to
core of proteins is called
enhancer sequences allows
an octamer.
18. The appearance of
for tissue specific
expression of genes if the
chromatin DNA is that of
enhancer proteins
beads on a string, with the octamer wrapped with DNA
themselves are expressed tissue specifically. Enhancer proteins
composing the beads and the DNA strand coated with histone
help to "clear" out the histones from a region of a chromosome
H1 (and H5) composing the string.
to allow transcription to occur.

363
The Antarctic fishermen still

Report that they quite get a thrill
Their common response?
They feel like James Bonds
Cuz theyve got a license to krill
22. Nuclear hormone
here as the same thing), with a part of the molecule extending
receptors, such as the
into the region of the protein that normally binds to co-
estrogen receptor, have DNA
activators. Thus, tamoxifen acts by stopping recruitment by the
binding domains and ligand
receptor of co-activators. Tamoxifen is used to treat tumors
binding domains. The
that are stimulated by the binding of estrogens to the receptor.
binding of the estradiol (and estrogen) ligand to the estrogen

receptor causes a conformational change in the protein, but
does not change the binding of the protein to DNA. Binding of
the estradiol DOES appear to activate the protein and thus
activate transcription of the genes that the
receptor binds to the promoter of.
23. The key to action of the nuclear hormone
receptor that binds estradiol is that binding of
estradiol favors binding of the receptor to co-
26. Altering chromatin structure is an essential function for

transcriptional activation in eukaryotes. Co-activator proteins
appear to play a role in this process by catalyzing the
acetylation of lysine residues in histones. Acetylation of histone
As the photon took o for his flight

He packed up his things really tight
When asked why he dragged
Only one Duel bag
He said he was traveling light
activator proteins. These co-activator proteins

help to turn on transcription of the relevant genes. Binding of
co-activator proteins by transcriptional factors, such as the
estrogen receptor is called recruitment.
24. An antagonist of the estrogen receptor is the drug tamoxifen.
Antagonists bind proteins and prevent them from responding to
their normal ligands. Binding of tamoxifen by the estrogen
receptor stops the receptor from activating transcription of
genes that it normally activates.
25. Tamoxifen appears to act by binding the estrogen receptor (I
use the terms estrogen receptor and nuclear hormone receptor
lysines neutralizes their positive charge,

changing the anity of histones for DNA
and changing the nature of their
interaction with DNA, thus allowing more
proteins to be able to gain access to the
DNA where the acetylation has occurred.
27. Proteins involved in transcriptional control often have

bromodomains. These regions of protein recognize and bind to
acetylated lysine residues in histones.
28. Altering chromatin structure involves a process called
remodeling. Steps in this process include 1) binding of a
transcription factor
to a promoter
sequence; 2)
recruitment of coactivators; 3)
Although candle makers protested

The decision could not be contested
Its price is quite steep
If at work they should sleep
There will not be a wick for the rested
364
acetylation of histone lysines by co-activators; 4) binding of the
The Vision Thing
To the tune of Star Spangled Banner
Did you know you can see

In the dimmest of light
With your rods and your cones
And their retinaldehyde
Found in rhodopsin its

Got a bond shaped as cis
But it changes its state
When a photon gets it straight
Then the signling kicks in

Thanks to a transducin
Cuz its GTP ways
Turn on diesterase
So gated ion channels stop

Charges from passing through
Such as sod-i-um plus one
And cal-ci-um plus two
'remodeling engine' at the acetylation site; 5) exposing of DNA

by the remodeling engine; and 6) binding of RNA polymerase II
to the exposed DNA.
29. Attenuation is a regulatory mechanism for several E. coli
operons involved in amino acid metabolism.
30. The tryptophan operon of E. coli (trp operon) controls the
expression of genes necessary to make tryptophan. When
tryptophan is abundant in E. coli, the ribosome moves quickly
along the trp operon mRNA, making protein. Control of the
operon is set up so that transcription of the entire operon only
occurs when tryptophan is limiting in the cells. When
tryptophan is abundant, transcription terminations very early.
The system is set up such that translation of the operon plays a
role in the early transcriptional termination.
31. In eukaryotic cells, the ferritin mRNA has a region of it called
an iron response element that can be bound by a protein called
IRE-BP (iron response element binding protein).
32. IRE-BP binds the iron response element when iron is absent. If
IRE-BP is NOT
bound to the iron

response
element (high
iron conditions),
Little Johnny the smartest debater

Pondered which integer was greater
Finding answers demands
That he count on his hands
Such is his digital calculator
365
ferritin is made because the IRE-BP does not block the

ribosome from translating the mRNA.
33. Thus, when iron concentration is high, ferritin is synthesized
to hold it. When IRE-BP is bound to the iron response element
(low iron conditions), ferritin is not made. So when iron is not
available, ferritin is not made. Gene expression of ferritin is
therefore a function of translational control.
34. The transferrin receptor has multiple iron response elements
at the 3' end of its mRNA. When IRE-BP binds to it, the 3' end
is protected and transferrin receptor is made.
35. So, when iron is low, the IRE-BP binds the transferrin receptor
mRNA, protecting it, and the transferrin receptor protein is
made to bring iron into the cells. When iron is high, the IRE-BP
leaves the mRNA's 3' end, leaving it susceptible to
degradation. Gene expression of the transferrin receptor is
therefore a function of stability of the gene's mRNA.
36. When iron inside the cell is high, ferritin is made to hold onto it
and when iron is low inside the cell, transferrin receptor is
made to bring more iron in.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
366
Section 3
Appendix - Exams
SECTION III
Exams we have given our students are
included in this section. They are hyperlinked
below.
1. Mid-Level Course #1
1. Exam 1 / Exam Key
Exam 1 (BB 450/550)

Section I Short answer - The questions below can generally be answered in 15
words or less. While you will not be required to use 15 words or less, excessively
long answers will be scrutinized closely. Each correctly filled in blank below will be
awarded three points, except as noted.

3. Final / Exam Key
2. Mid-Level Course #2
3. Final / Exam Key
3. Basic Course Exams
Important items
A. The logarithm of a number greater than one is a positive number.
B. The logarithm of a number less than one is a negative number
C. For any number X (except X=0), ln (X) = -ln(1/X)
[or log(x) = -log(1/x)]
D. pH = pKa + log {[Salt]/[Acid]}
E. pH = -log[H+] , pOH = -log[OH-], pKa = -Log[Ka], pH + pOH = 14
F. pKa values to assume for amino acids alpha amine = 10, alpha carboxyl =

2.2, R-group amine = 12, R-group carboxyl = 4.4, R-group sulfhydryl = 8
G. G = G + RTLn([Prod]/[React])
H. R (the gas constant) = 8.3x10-3 kJ/Kmol

1. You have an acetate buer. You add sodium acetate (salt) to it. What happens
to the pH?

4. Final / Exam Key
2. You are examining a 2D gel. What was the name of the first technique that was
used to perform this technique?
367
3. You want to make the smallest polypeptide you can that will
you would use to calculate Km (2 points) and tell precisely how
have the maximum positive charge without using the same amino
you would calculate Km from the value of this point (2 points).
acid twice. Give a sequence of such a polypeptide that you would

make
4. Name all of the amino acids that cannot exist as zwitterions
5. You have an acetate buer. You manage to magically destroy
half of the sodium acetate in it without converting it into anything
else. What happens to the pH of the resulting solution?
6. Tell precisely/completely what one joins together to make a
peptide bond. Structures not necessary or desired.
11. Velocity of a car is given in miles per hour. How is the velocity
of an enzymatic reaction given? (must be precise)
7. Scurvy arises as a result of the structural weakness of what

compound in the body?
12. What is the name of the technique described in class uses

beads with tiny tunnels in them?
8. Name the categories described in class that the following

amino acids are grouped in
13. Name all of the levels/types of protein structure stabilized at

least partly by hydrogen bonds
Leucine =
Section 2 Calculations - For each of the problems in this
Cysteine =
Asparagine =
9. What chemical named in class can one use to reduce a
covalent bond between two cysteine side chains?
10. A scientist studies the kinetic behavior of an enzyme and
section, ORGANIZE and LABEL your calculations clearly. No

partial credit will be given without clearly labeled calculations.
1. You have 1L of a 0.6M buer at maximum buering capacity.
When you add 0.1 moles of HCl to the buer (no volume change),
the pH is 9.4. Show how you would calculate the pKa of the
buer.
obtains the following plot. Clearly mark the point on the graph
368
2. A scientist has the polypeptide shown below at pH = 0 and
both statements can be correct, depending on how you calculate
begins adding NaOH to it.
Kcat. What is the head of the lab thinking? (10 points)
glycine-arginine-leucine-aspartic acid-methionine-leucine
a. Using the pKa values from the first page of the exam,
draw the titration curve (with properly labeled axes) for this
molecule up to pH = 14.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
b. Indicate the charge on the polypeptide at each relevant

place on the graph
c. Show how to calculate the pI of this polypeptide
Section 3 Explanations For each question, provide a brief
explanation of the phenomenon. Long rambling answers that are
not to the point will lose points, even if they contain part of the
correct answer.
1. I named at least four molecules/atoms/ions that aect
hemoglobins structure upon binding to it. Name each molecule/
atom/ion and describe precisely where it binds on hemoglobin
and how it aects oxygen anity (20 points)
2. Your friend is studying an enzyme in the presence of a noncompetitive inhibitor and says that it gives a value of Kcat dierent
than the same enzyme without the non-competitive inhibitor.
Another friend says that it should have the same Kcat as the
uninhibited reaction. The head of the lab walks up and says that
369
Exam 2 (BB 450/550)

Section I Short answer - The questions below can generally
be answered in 15 words or less. While you will not be required to
use 15 words or less, excessively long answers will be scrutinized
4. Only one of the allosteric eectors of ATCase favors the Tstate. Which one?
5. Draw the structure of the first glycolysis intermediate
containing two phosphates.
closely. Each correctly filled in blank below will be awarded three
6. Precisely what chemical reaction on what molecule/compound/
points, except as noted.
enzyme does warfarin directly inhibit (four points)?
Important items

1. What is the dierence between the DNA sequence recognized

by a restriction enzyme and the DNA sequence recognized by its
corresponding methylase?
2. Carbonic anhydrase is more active at pH 8.5 than it is at pH 7.
Precisely what is favored at the higher pH that causes this?
3. I talked about at least three dierent ions that could act as
7. Define the term anomers

8. Draw the Fischer structure of and name the only pentose you
are responsible for knowing the structure of (no partial credit)
9. What is the name of the only enzyme of glycolysis that
catalyzes a redox reaction?
10. In the catalytic action of serine proteases, what must happen
to move the catalytic triad closer together to create the alkoxide
ion and start the catalytic process?
11. Where in the cell are O-linked glycoproteins made?
nucleophiles in enzyme catalysis. Draw three of these (hint be
12. What are the name(s) of the second/third messenger(s) for the
sure to draw the form of each ion that acts as the nucleophile)
pathway involving phospholipase C?
370
13. Name a compound described in class that is an inhibitor of
BCR-ABL

correct answer.
partial credit will be given without clearly labeled calculations. (15
1. You have four friends. Two are born without alpha-1-
points each)
antitrypsin and two are born with a normal alpha-1-antitrypsin.
1. A reaction is at equilibrium at a temperature of 300K with a

[Reactant] = .4M and [Product] = 1.0M. Using as much of this
information as possible, write an equation to calculate the G
when there are equal amounts of the two (15 points)
Their circumstances are below:

A.
B.
C.
D.
No alpha-1-antitrypsin - smokes
No alpha-1-antitrypsin - doesnt smoke
Regular alpha-1-antitrypsin - smokes
Regular alpha-1-antitrypsin - doesnt smoke
2. The reaction below meets the needs of the bodys muscles

remarkably well. Explain how the G for this reaction varies
according to whether one is exercising or resting and how that
Predict the relative tendency to have emphysema you would
provides the muscles with what they need to keep the body
expect for each of the friends in the comparison below based on
functioning. (15 points)
class discussion. Justify your answer at the end of the problem.

(15 points)

a. Compare A to B (is emphysema in A > B, A<B, or A=B)
(G = + 12 kJ/mol)
b. Compare C to D (is emphysema in C > D, C<D, or C=D

c. Compare B to D (is emphysema in B > D, B<D, or B=D)
371
2. You are walking home one night when out of the bushes jumps
your biochemistry professor singing loudly and very o key. This
scares you terribly, so you start secreting the same hormone you
would if a grizzly bear were chasing you. Name the hormone and
show/describe the entire signaling pathway described in class
that it activates. (15 points)
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
372
Final Exam (BB 450/550)
5. Name three forces stabilizing quaternary interactions
6. A deficiency of what dietary molecule causes scurvy?
7. Name the only molecule described in class that competes with
oxygen for the binding site on hemoglobin

8. To what atom does oxygen bind in hemoglobin?
Important items

9. Precisely where on ATCase does aspartate bind?

10. What is the name of the enzyme in glycolysis that produces
an intermediate that aects hemoglobins anity for oxygen?
11. Name the only enzyme in glycolysis that is regulated both
allosterically and by covalent modification
1. One method of separating proteins described in class
12. Draw the Haworth structure for the only hexose given in class
combines two dierent separation techniques. Name the overall
for which you must know the furanose form
method and the names of the two separation techniques

2. Name all of the amino acids described in class that can be
phosphorylated
13. What is the name of the enzyme that catalyzes the

breakdown of blood clots?
3. Describe or illustrate (precisely) below the reaction that
to move the catalytic triad closer together to create the alkoxide
mercaptoethanol catalyzes
ion and start the catalytic process?
15. Gleevec inhibits Bcr-Abl. What enzymatic reaction does this
protein catalyze?
373
16. What bond is broken by the nucleophilic attack in the fast
24. Name the only enzyme I described in class that is used both
step of the catalytic mechanism of serine proteases?
in the breakdown of glycogen pathway and the synthesis of
17. Name three first messengers described in class
glycogen pathway
25. What does it take to convert glycogen phosphorylase a from
the T state to the R state?
18. A friend tells you that a new buer has been discovered in
which there is more salt than acid when the pH is the same as the
pKa. Using math, explain why this is true or false. (Your answer
must also say TRUE or FALSE)
19. Kevin Aherns hypothesis of why Americans are becoming

more and more obese says it is due to the bypassing of an
important enzymatic reaction. What is the name of this enzyme?
20. What molecule is synthesized in animals in order to make
NAD when oxygen levels are low?
21. In utilization of galactose, it is first phosphorylated and added
to a molecule that results in the release of glucose-1-phosphate.
To what molecule is the phosphorylated galactose attached?
22. Name a transcription factor made/activated in cells when
oxygen is lacking.
23. Name a molecule that activates an enzyme in feed-forward
activation.

points each)
1. You have 1L of a 0.6M buer with twice as much salt as acid.
After you add .1 moles of HCl (no volume change) and raise the
temperature of the solution by 10 degrees, the pH is 8.32. Write
an equation to calculate the pH before the HCl was added.
2. A reaction starts with 5 times as much reactant as product,
but at equilibrium, it only has 3 times as much reactant as
product.
a. Write an equation to show how to calculate G (3 points)
b. Write an equation to show how to calculate G at the very
beginning (3 points)
c. Show how you would determine the sign of the G (3
points)
374
d. What was the sign of G at the start of the reaction?

Why? (3 points)
3. An analysis of an enzymatic reaction is performed in the
presence and absence of an inhibitor. The inhibitor appears to
reduce the Vmax of the enzyme.
a. Draw a single Lineweaver Burk plot showing the
uninhibited and the inhibited reaction. Clearly label all
relevant features on the graph. (5 points)
b. Draw a single V vs. [S] plot for the showing the uninhibited
and the inhibited reaction. Clearly label all relevant features
on the graph. (5 points)
c. What type of inhibition is occurring? (2 points)
c. Name the most important allosteric regulator of the two

pathways and show precisely how its synthesis and breakdown is
regulated. (6 points)
2. Glycogen phosphorylase is regulated allosterically and by an
enzyme it carries around with it. Draw a scheme illustrating all of
the ways in which glycogen phosphorylase is regulated and also
a scheme showing how the enzyme it carries can aect the
enzyme. (10 points total)
3. We have talked this term about three broad ways in which
enzyme activity can be regulated. Name each way. Name one
enzyme regulated each way. Give detail as appropriate for each
means of regulation. If external molecules or modifications are
relevant, your answer must include them and a description must
include the eect the molecules or modifications have on each
enzyme. (12 points)
correct answer.
1. Regulation of glycolysis and gluconeogenesis is complex. It
Jump to Chapter
involves allosteric interactions, covalent modifications, and

control of enzymes that make/break down moleules that aect
the pathway.
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
a. Name the enzymes regulated in each pathway (7 points)

b. Name one allosteric activator and one allosteric inhibitor of
each enzyme that is reciprocally regulated (4 points)
375
Exam 1 (BB 451/551)

Section I: (20 points total) The statements in this section can be
completed by any of the lettered responses following it. Each
statement may have more than one answer that is correct, one
answer that is correct, or no answers that are correct. Students
should clearly circle only those responses that complete the
sentence to make a correct statement. Points will be awarded for
each circled response that makes a correct statement and for
each uncircled response that makes an incorrect statement. For
example, the practice question below has three correct answers
(b,c,d). You would receive four points if you circled b,c, and
d. You would receive one point if you circled a and b. You
would receive no points if you circled only a. If we have
uncertainty about whether or not an answer is marked, it will
automatically be counted as a wrongly answered question. Be
clear in your markings.
Practice question #A: Oregon State University

A.
B.
C.
D.
is a Peruvian factory
is located in Corvallis, Oregon
has a mascot named Benny Beaver
has students from all over the world.
2. With respect to respiratory control,

A. cyanide will stop oxidative phosphorylation first

B. exercising will favor production of NAD+

C. resting will favor decrease in ADP
D. 2,4 DNP (miracle diet drug) will increase NADH and

temperature
3. With respect to membrane transport,

A. lactose permease is an anti-port

B. it requires ATP

C. it requires diusion
D. it cannot be electroneutral if sodium is involved
4. With respect to electron transport,

A. electrons get pumped into the intermembrane space

B. oxygen is the terminal proton acceptor

C. the Q cycle occurs in Complex III
D. 2,4 DNP will increase oxygen consumption
5. In tightly coupled mitochondria,

A. increasing oxygen will increase the proton gradient

B. lack of NAD will ultimately reduce electron flow

C. uncoupling protein (UCP) will reduce ATP production
D. the citric acid cycle will run fastest when electron
transport runs fastest
1. With respect to the citric acid cycle,

A. succinate dehydrogenase requires FAD for catalysis

B. it includes two decarboxylations not found in the

glyoxylate cycle

C. NADH is not used

D. it uses oxygen in a one of its enzymatic reactions
376
Section II: (50 points total) Each sentence below in this

section is missing a word or phrase to complete it. Fill in the
blank as appropriate to complete the sentence with a correct
statement. Each correctly filled in blank below will be
awarded two points.
1. Name the five carbon intermediate in the citric acid cycle
_______________________
2. Which enzyme of the citric acid cycle catalyzes a reaction that
pulls a reaction preceding it? _______________________
9. If one membrane protein pump were missing from the cell,

neural transmission as described in class would not be possible.
What pump is that? _______________________
10. What is the basis for exclusion of potassium from a sodium
channel? _______________________
11. What is the basis for exclusion of sodium from a potassium
channel? _______________________
3. Which enzyme of the citric acid cycle requires lipoamide?

_______________________
12. Active transport always has at least one mechanistic

dierence from passive transport. It is the reason that active
transport requires energy. What is that dierence?
_______________________
4. Which enzyme of the citric acid cycle catalyzes the only

oxidation that doesnt produce NADH? _______________________
13. What is the driving force for passive transport?

_______________________.
5. What citric acid cycle intermediate can be readily made from

glutamic acid in an anaplerotic reaction?
_______________________
14. What is the definition of a symport?

_______________________.
6. How many oxaloacetates are produced per turn of the citric

acid cycle? _______________________
15. You remove digitoxigenin from a heart cell that had been
treated with it. What happens to the concentration of sodium
outside of the cell? _______________________.
7. Name a poison described in class that reacts with a citric acid

cycle coenzyme _______________________
16. In the Q cycle, what takes electrons away from Complex III?
_______________________.
8. Which class of membrane-related proteins would be the most

likely to have hydrophobic amino acids on its exterior?
_______________________
17. Complex V has a rotating complex that make ATP. It has

three dierent configurations. What is the form of the
configuration where ATP is released? _______________________.
18. You treat a mitochondrion 2,4 DNP and cyanide. What
happens to oxygen consumption? _______________________.
377
19. A mitochondrion is treated with 2,4 dinitrophenol (2,4 DNP).

What happens to its oxygen consumption?
_______________________.
20. The mitochondria in a cell are tightly coupled. The citric acid
cycle is running furiously. What normal explanation (not exotic or
unusual) makes the most sense about what this cell is doing to
cause this? _______________________.
21. If one wanted to make a photosynthetic fish, as described in
class, what protein would you need to do it?
_______________________.
22. Describe the numbering system for an omega fatty acid.

Which carbon is number one? _______________________.
23. What is the name of the category of ATP-using transport
proteins that involve a phosphoaspartate?
_______________________.
24. What is the charge on a copper atom in Complex IV after it
has accepted an electron? _______________________.
Section III: (30 points total) Matching.
____ 1. Na/K ATPase

____ 2. Isocitrate

____ 3. Brown fat

____ 4. Acetaldehyde

____ 5. Decarboxylation

____ 6. Sphingomyelin

____ 7. Malate synthase

____ 8. Antimycin A

____ 9. Oxygen

____ 10. ADP

____ 11. H2O2

____ 12. Coenzyme Q

____ 13. Synaptic vesicles

____ 14. Malate

____ 15. Thiamine pyrophosphate (TPP)

A. Can stop oxygen consumption indirectly

B. Limiting for people not exercising
C. Produced by superoxide dismutase
D. Glyoxylate cycle requirement
E. Carries electrons in pairs
F. Five carbon intermediate
G. Phosphorylated membrane component
H. Neurotransmitter described in class
I. Neurotransmitter holder
J. Complex IV inhibitor
K. Superoxide dismutase product
L. Process that does not occur in glyoxylate cycle
M. Six carbon intermediate
N. Symport
O. Blocked by tetrodotoxin
P. Produced by citrate synthase
Q. Gets oxidized after being shuttled
R. Ethanol precursor
S. Produced by oxidative phoshorylation
T. Electro-genic
U. Can become uncoupled
V. Carries a two carbon group
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
25. If you wanted to raise the Tm of a membrane, how would you

alter the chemical composition of its fatty acids?
_______________________.
378
Exam 2 (BB 451/551)

sentence to make a correct statement. Points will be awarded
for each circled response that makes a correct statement and for

A.
B.
C.
D.
has students from all over the world
1. With respect to cholesterol metabolism,

A. it uses palmitoyl-CoA and serine

B. it includes an intermediate of squalene

C. ATP is not used
D. it is a precursor to making vitamin D, bile acids, and

steroid hormones
2. With respect to fatty acid metabolism,

A. it starts with oxidation of medium chain fatty acids in

peroxisomes
B. oxidation of saturated fatty acids involves cis

intermediates

C. enoyl-CoA isomerase is not needed for oxidation of

saturated fatty acids
D. ketone bodies use the last enzyme of fatty acid

synthesis
3. With respect to prostaglandins,

A. ibuprofen is a COX-1 inhibitor

B. steroids inhibit COX-2 enzymes
C. they can be produced from thromboxanes or

leukotrienes

D. PLA-2 inhibits the action of aspirin
4. With respect to nucleotide metabolism,
A. deoxyribonucleotides are made from

ribonucleoside diphosphates
B. PRPP amidotransferase balances relative amounts of

purines

C. NDP Kinase (NDPK) converts ADP to dADP

D. dUTPase converts dUTP to dTTP
5. With respect to DNA replication,
A. the beta clamp is responsible for making DNA

Polymerase III more processive

B. helicase forms double helices at the rate of 6000 rpm

C. Okazaki fragments are only made in the 5 to 3 direction

D. lagging strand replication occurs in the 3 to 5 direction
379
Section II: (50 points total) Each sentence below in this section
is missing a word or phrase to complete it. Fill in the blank as
appropriate to complete the sentence with a correct statement.
Each correctly filled in blank below will be awarded two points.
1. What is the name of the transcription factor that helps control
synthesis of HMG-CoA reductase? _______________________
2. I named four general mechanisms for regulating HMG-CoA
reductase. Name three of them (1 point each)
_______________________, _______________________,
_______________________
9. Describe accurately in words what enoyl-CoA isomerase

catalyzes
10. Name the enzyme that catalyzes the first reaction in the
pathway leading to ketone bodies _______________________
11. Name the molecule produced by catalysis by the only
regulated enzyme of fatty acid biosynthesis
_______________________
12. Name the molecule that shuttles the precursor of fatty acid
synthesis to the cytoplasm _______________________
3. What group of molecules has their synthesis stopped by

aromatase inhibitors? _______________________
13. How does carbamoyl phosphate synthetase protect its

intermediate product from water? _______________________.
4. I mentioned three factors to consider for controlling the level of

cholesterol in the body. Name them (1 point each)
_______________________, _______________________,
_______________________
14. What is the name of the enzyme that balances purines and
pyrimidines in de novo ribonucleotide synthesis?
_______________________.
5. What is the name of the cellular structure missing in familial

hypercholesterolemia? _______________________
6. What is the name of the last product of beta oxidation of a
fatty acid with an odd number of carbons?
_______________________ (name must be precise)
7. Name the only regulated enzyme in either fat or fatty acid
breakdown _______________________
8. Draw the structure that the enzyme thiolase would cut
_______________________
15. What molecule is the energy source for synthesis of AMP?

_______________________.
16. What is the name of the molecule that is the branch point in
de novo purine ribonucleotide synthesis?
_______________________.
17. Write out the reaction catalyzed by adenylate kinase
_______________________.
18. What atom does RNR (ribonucleotide reductase) catalyze the
removal of? _______________________.
380
19. What is the name of the electron source for fatty acid
biosynthesis? _______________________.
Section III: (30 points total) This section of the exam is on the
next page.
20. What is the name of the first electron acceptor in fatty acid
oxidation? _______________________.
21. Name the form of DNA discovered by Rosalind Franklin
_______________________.
22. Name the enzyme necessary for joining the DNAs of Okazaki
fragments _______________________
22. What is the name of the first protein to bind to the E. coli
DNA replication origin? _______________________.
23. Precisely what does telomerase use as a template to make
telomeres? _______________________.
24. What enzymatic activity is present in DNA polymerase I and
III, but lacking in reverse trancriptase of HIV?
_______________________.
25. Give the ratio that determines whether or not a DNA is
relaxed or not (note I am looking for the units, not a number)
_______________________.
381
Matching
Each term on the left has a phrase or term on the right which best describes or matches it.
Place the letter of the term/phrase on the right in the blank before the term on the left that
it best matches. Only one letter is appropriate in the blank. Note that there are more
terms on the right than there are blanks, so not every term on the right has a best match.
Terms on the right may be used once, more than once, or not at all. If we cannot read your
writing or if you put two letters in any blank on the left, your answer will be counted wrong
automatically. Each correctly matched pair is worth two points.
____ 1. dnaB

____ 2. Guanosine

____ 3. Gyrase

____ 4. Arachidonic acid

____ 5. LDL

____ 6. Serine

____ 7. CDP

____ 8. Cholesterol

____ 9. dATP

____ 10. S-Adenosyl Methionine (SAM)
____ 11. HMG-CoA

____ 12. Lipase

____ 13. Ganglioside

____ 14. ACP

____ 15. Glycerol

A. Necessary for fat digestion

B. Lipoprotein complex
C. Palmitate precursor
D. Requires vitamin B12
E. Replaces CoA of malonyl-CoA
F. Feedback inhibitor
G. ATCase inhibitor
H. Lacking in Lesch-Nyhan syndrome
I. Leukotriene precursor
J. Sphingolipid
K. Prostaglandin product
L. Topoisomerase
M. Nucleoside
N. Energy source in activated intermediates
O. Branch point molecule for two pathways
P. Necessary for sphingolipid synthesis
Q. Glycerophospholipid
R. Methyl donor
S. INSIG target
T. Can be made into glucose
U. Helicase
V. Allosteric regulator of ribonucleotide

reductase
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
382
Final Exam (BB 451/551)

Section I: (40 points total) The statements in this section can be completed
by any of the lettered responses following it. Each statement may have
more than one answer that is correct, one answer that is correct, or no answers that are correct. Students should clearly circle only those responses
that complete the sentence to make a correct statement. Points will be
awarded for each circled response that makes a correct statement and for
each uncircled response that makes an incorrect statement. For example,
the practice question below has three correct answers (b,c,d). You would
receive four points if you circled b,c, and d. You would receive one
point if you circled a and b. You would receive no points if you circled
only a. If we have uncertainty about whether or not an answer is marked,
it will automatically be counted as a wrongly answered question. Be clear
in your markings.
!
A. is a Peruvian factory
!
B. is located in Corvallis, Oregon
!
C. has a mascot named Benny Beaver
!
D. has students from all over the world.
!
A. GDP is not required
!
B. lipoic acid is not used
!
C. oxygen is not a substrate in the reactions of the cycle
!
D. oxidative decarboxylation does not occur
2. With respect to movement of compounds across biological membranes,
!
A. active transport requires ATP
!
B. water cannot readily cross them
!
C. a proton gradient is required
!
D. protons generally require a protein to move across them

A. oxidative phosphorylation will speed up to make up for an
!
!
oxygen deficit
B. low ADP levels will have the same overall effect as low oxygen
!
!
levels
!
C. increasing intermembrane space pH will reduce ATP production
!
D. cyanide will reduce both ATP production and the proton gradient
4. With respect to the Q cycle,
A. it explains how electrons come in pairs, but are converted to
!
!
moving singly
!
B. it involves coenzyme Q in three different forms
!
C. Cytochrome C carries electrons to complex III
!
D. each QH2 sends electrons to two different things
5. With respect to fatty acid oxidation,
!
A. it occurs in the cytoplasm and the peroxisomes
!
B. it produces FADH2 and NADH
!
C. it only occurs on fatty acids shorter than 16 carbons
!
D. it occurs when the fatty acid is bound to ACP
6. With respect to fatty acid synthesis,
!
A. it is allosterically activated by palmitate/palmitoyl-CoA
!
B. it occurs in the cytoplasm and endoplasmic reticulum
!
C. it uses malonyl-ACP
!
D. it requires a decarboxylation
!
A. aspirin is a COX inhibitor
B. inhibiting phospholipase A2 will stop release of fatty acids
!
!
from membranes
!
C. they are precursors of cholesterol
!
D. they are pain relievers
!
A. it uses ribonucleotide reductase to convert all dNDPs to dNTPs
!
B. it uses CTP to make UTP
!
C. it uses dTMP to make dUMP
!
D. it uses DHFR to make dNDPs from NDPs
383
6. Name one enzyme unique to the glyoxylate cycle

9. With respect to transcription,
!
A. it occurs at a structure called a replication fork
!
B. the template strand is complementary to the one being made
!
C. it makes RNA in the 3 to 5 direction
!
D. it requires a ribosome
_______________________
7. You take a heart cell that was treated with digitoxigenin and remove the
digitoxigenin. What happens to the concentration of calcium outside of the
10. With respect to gene expression,

!
A. attenuation involves early transcriptional termination
!
B. the lac repressor binds to DNA when it binds allo-lactose
!
C. CAP binds to DNA when it binds to cAMP
!
D. IRE-BP binds to DNA when it does not bind iron
cell? _______________________.
8. Name a specific sphingolipid named in class that contains a phosphate

_______________________
Section II: (80 points total) Each sentence below in this section is missing a
word or phrase to complete it. Fill in the blank as appropriate to complete
the sentence with a correct statement. Each correctly filled in blank below
will be awarded two points.
1. What coenzyme named in class does arsenate react with?
_______________________
2. Which enzyme of the citric acid cycle catalyzes a reaction that produces
FADH2? _______________________
3. What is the definition of an antiport? _______________________.
9. Name the class of lipid described in class that contains complex carbohydrates _______________________
10. A gradient of which ion is used as an energy source to remove calcium

ions from heart cells? _______________________.
11. What is the criterion we use to determine if a transport mechanism is

active versus passive? _______________________.
12. In what class of membrane transporters does phosphoaspartate appear?

_______________________.
4. Which enzyme of the citric acid cycle catalyzes a reaction that pulls the
one preceding it? _______________________
13. A cell (and its mitochondria) are treated with 2,4 dinitrophenol (2,4
DNP). What happens to the citric acid cycle in this cell?
5. What amino acid is alpha-keto-glutarate readily converted to in an
_______________________.
anaplerotic reaction? _______________________
384
14. Name one of the two isoprenes described in class
22. Name the enzyme that catalyzes conversion of fatty acids with a cis dou-
_______________________.
ble bond between carbons 3/4 to a trans double bond between carbons 2/3
_______________________.
15. Complex V has a rotating complex that makes ATP. It has three different
configurations. What is the form of the configuration where ATP is re-
23. What causes gout? _______________________.
leased? _______________________.
24. Where does one find acyl-CoA dehydrogenases that act on fatty acids of
16. Describe precisely the catalytic activity of DNA Polymerase I that re-
20 carbons? _______________________.
moves RNA primers _______________________.
17. Precisely what does telomerase copy to make a telomere?
25. Name a covalent modification of an enzyme in fatty acid metabolism
_______________________.
and the effect of that covalent modification

Enzyme modified = _______________________.
18. How does one completely inhibit PRPP amidotransferase?
Modification = _______________________.
_______________________.
Effect of modification _______________________.
19. What is the name of the molecules that donate single carbons in nucleo-
26. Name the enzyme used in DNA footprinting _______________________.
tide metabolism (hint Im not looking for carbon dioxide or bicarbonate) ?

_______________________.
27. Name the specific catalytic entity that forms phosphodiester bonds between ribonucleotides _______________________.
20. What molecule does aspirin prevent from being acted on by COX?
_______________________.
28. Name the specific catalytic entity that forms peptide bonds between
amino acids in bacteria _______________________.
21. In fatty acid synthesis, describe the chemical step after deyhdration (all I
need is a name describing the chemical reaction) _______________________.
29. Define the term template strand _______________________.
385
30. Name the protein described in class that spins at 6000 rpm
_______________________.
31. In attenuation of the trp operon, what is the function of the structure in
the mRNA that only forms when there is abundant tryptophan in the cell?
Section III: (30 points total) Matching. Each term on the left has a phrase or term
on the right which best describes or matches it. Place the letter of the term/phrase
on the right in the blank before the term on the left that it best matches. Only one
letter is appropriate in the blank. Note that there are more terms on the right than
there are blanks, so not every term on the right has a best match. Terms on the right
may be used once, more than once, or not at all. If we cannot read your writing or if
you put two letters in any blank on the left, your answer will be counted wrong automatically. Each correctly matched pair is worth two points.
_______________________.
32. What does tamoxifen prevent the estrogen receptor from interacting
with? _______________________.
33. In elongation, which direction is the ribosome moving relative to the

mRNA? _______________________.
34. Name the 7TM of vision that is bound to Vitamin A

_______________________.
35. Name the G protein involved in the sense of smell

_______________________.
36. Describe the unusual bond in a lariat structure

_______________________.
37. Describe what rho does and how it does it _______________________.
____ 1. TPP! !
!
!
____ 2. Intron!
!
!
____ 3. Transducin! !
!
____ 4. Gyrase!
!
!
____ 5. dATP!!
!
!
____ 6. Inducer!
!
!
____ 7. Thiolase!
!
!
____ 8. Statin!
!
!
____ 9. Active transporter! !
____ 10. Tamoxifen! !
!
____ 11. Trans fat! !
!
____ 12. Okazaki fragments!
____ 13. Carnitine! !
!
!
!
!
!
!
____ 14. Anti-codon!!
!
____ 15. Ribozyme! !
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
A. Peptide bond catalyst(s)

B. Purine breakdown product(s)
C. Binds retinal
D. Hydrogenation byproduct(s)
E. tRNA component(s)
F. Sphingolipid(s)
G. Lagging strand synthesis product(s)
H. IPTG
I. G protein(s)
J. Ketone body enzyme(s)
K. Prostaglandin enzyme(s)
L. Na/K ATPase
M. Pyruvate dehydrogenase complex
!
component(s)
N. pre-mRNA component(s)
O. Reverse transcriptase(s)
P. Topoisomerase(s)
Q. Helicase(s)
R. Translation chain terminator(s)
S. Copper
T. Mitochondrial membrane crosser(s)
U. Ribonucleotide reductase inhibitor(s)
V. HMG-CoA reductase inhibitor
W. Fatty acid synthase end product(s)
X. Estrogen receptor binder(s)
38. Precisely where is the wobble base of the anticodon found ?

_______________________.
386
Exam 1 (BB 350)

Section I: The statements in this section can be completed by
any of the lettered responses following it. Each statement may
have more than one answer that is correct, one answer that is
correct, or no answers that are correct. Students should clearly
circle only those responses that complete the sentence to make a
correct statement. Points will be awarded for each circled
response that makes a correct statement and for each uncircled
response that makes an incorrect statement. For example, the
practice question below has three correct answers (b,c,d). You
would receive eight points if you circled b,c, and d. You
would receive two points if you circled a and b. You would
receive no points if you circled only a. If we have uncertainty
about whether or not an answer is marked, it will automatically be
counted as a wrongly answered question. Be clear in your
markings.

A. is a factory in Portland


1. With respect to hydrogen bonds,

A. they are weaker than covalent bonds

B. they are weaker than disulfide bonds

C. they are what make peptide bonds

D. water does not have to be involved
2. With respect to amino acids,

A. glycines R group is a hydrogen

B. lysines R group is more hydrophobic than leucines

C. methionine is the only one with a sulfur in the R group

D. every one of them can exist as a zwitterion
3. With respect to enzymes,

A. Km is a velocity

B. Vmax/2 is the same as Km

C. Kcat varies with substrate concentration

D. Kcat = Km /[enzyme]
4. With respect to information in the syllabus,

A. there is no fixed grading scale

B. grades are curved

C. the grading scale is 90/80/70/60
Section II: 1. Each sentence below in this section is missing a

word or short phrase to complete it. Fill in the blank as
Each correctly filled in blank below will be awarded three points.
You MUST be precise in your definitions.
1. Describe the dierence between a strong acid and a weak

acid. ________________
2. Name an amino acid that makes disulfide bonds
________________
3. How does a fibrous protein dier from a globular protein?
________________
4. A solution has a pH of 6. Write an equation for its hydroxide
ion concentration ________________
5. What is the name of the group in myoglobin that contains the
iron? ________________
387
6. Define allosterism ________________
Section III Problems/Long Answer
7. You have a buer solution with more salt than acid and the pH
1. Explain at the molecular level why smokers breathe more
is 6. What can you say about the pKa? ________________
heavily when exercising than non-smokers. (16 points)
8. Explain from an energetic perspective how enzyme catalysis

works (one sentence should suce).
9. What units does KM have? ________________
10. Name three separation techniques described in class
2. An exotic amino acid is discovered that has two amine groups
11. What name do we give to the molecule(s) that an enzyme
and three carboxyl groups. The pKa values of the groups are
catalyzes a reaction on? ________________
below
Amino #1 = 8.8
Amino #2 = 12.1
Carboxyl #1 = 1.1
Carboxyl #2 = 4.2
Carboxyl #3 = 6.6
a. Draw and label CLEARLY a titration curve with all relevant
values for this amino acid (12 points)
b. Calculate the approximate charge this amino acid would have
at a pH of 5.5 (4 points)
388
3. For the following problem, ORGANIZE and LABEL your

calculations clearly. No partial credit will be given without clearly
labeled calculations. You do NOT have to enter the numbers into
a calculator. You simply need to show the last step before
punching it in.
You have a 500 ml of a .6M buer of ahernium (pKa = 9.11) in
which the concentration of acid is twice that of the salt.
a. Write an equation to calculate the pH of this buer (4 points)
b. If you wish to get the buer to maximum buering capacity,
describe whether you would use HCl or NaOH and how many
moles you would use. You will need to clearly show and label
your calculations to explain your answer. (16 points)
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
389
Exam 2 (BB 350)


markings.




1. With respect to fatty acids,

A. more unsauration lowers their melting temperatures

B. greater length raises their melting temperatures
C. oils have more unsaturated fatty acids than fats

D. fish membranes have more unsaturated fatty acids

than humans
2. With respect to nucleic acids,

A. adenine does not contain a sugar

B. nucleosides do not contain a sugar

C. nucleotides do not contain a phosphate

D. RNA contains ribose, but not deoxyribose
3. With respect to membranes,

A. anchored membrane proteins project through both sides

of the lipid bilayer

B. liposomes are not natural cellular membranes

C. carbon dioxide cannot cross the lipid bilayer

D. cholesterol raises the transition temperature of a

membrane
4. With respect to control of enzyme activity,

A. covalent modification always activates enzymes

B. allosterism always inactivates enzymes

C. feedback inhibition works allosterically

1. What molecule found in the membranes of cells widens the

cells transition temperatures (Tm values)? ________________
2. Define allosterism (no points for being close)
________________
390
3. ATCase has two dierent kinds of subunits. What category of
subunit does aspartate bind to? ________________

1. E. coli DNA polymerase has three essential activities for DNA
4. Carbohydrates are only found attached to the outside of cell
replication. Name and explain the enzymatic activity that each
membranes. What is their function, as described in class?
one catalyzes. (16 points)
________________
2. Draw out the central dogma and identify the exception to it
5. What is the driving force for the movement of molecules in
with respect to retroviruses, such as HIV
facilitated diusion? ________________

3. I described in class how E. coli can switch from making one
6. What three carbon molecule forms the backbone of
class of genes to making another class of genes, due to changes
glycerophospholipids, but not sphingolipids? ________________
of circumstances. Describe precisely what is involved in making
7. What is required for a membrane transport system to be

described as active transport? ________________
8. Name an inhibitor of prostaglandin synthesis
________________
9. What chemical change happens to vitamin A that results in
nerve signaling? ________________
10. What is the dierence between a deoxyribonucleotide and a
this switch (hint this has nothing to do with lactose).

4. The lac operon has controls that allow it to adapt to the cells
needs for energy. Explain all of the controls listed in class and
how they allow for this adaptation.
5. List and describe three modifications to eukaryotic mRNAs
that do not happen to prokaryotic mRNAs.
Jump to Chapter
deoxyribonucleoside (be precise in your answer)?

11. In attenuation of the trp operon, when there is sucient
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
tryptophan, what process is terminated? ________________
391
Exam 3 (BB 350)

markings.




1. With respect to tools for biotechnology,
A. a plasmid is a circular DNA that replicates in human cells
B. a polylinker is a short stretch of DNA in a plasmid with

multiple restriction sites

C. enzymes used to cut DNAs at specific places are called

methylases

D. a plasmid for making protein in a cell would have to have

a promoter
2. With respect to sugar structures,

A. a hexose cannot be a furanose
B. the Fischer straight chain structure of glucose has no

anomeric carbon
C. a reducing sugar is easily reduced

D. a glycoside always has a bond involving the anomeric

carbon
3. With respect to translation,

A. the 23s rRNA catalyzes the formation of peptide bonds

B. initiation does not involve any peptide bond formation

C. EF-Tu is involved in translocation

D. ATP is required for translocation
4. With respect to polysaccharides,

A. cellulose is a polymer of glucose
B. chitin is a polymer of fructose
C. bacterial cell walls contain peptidoglycan structures with

D-amino acids

Each correctly filled in blank below will be awarded three points,
except as noted. You MUST be precise in your definitions.
1. What is the name of the bacterial protein factor responsible for
translocation during translation? ________________
2. Precisely where is the wobble base of the anti-codon?
________________
392
3. Describe the physical appearance of a messenger RNA during

eukaryotic translation ________________
13. Draw the structure of L-glucose in the Fischer form

14. Draw the structure of alpha-D-ribose as a furanose
4. Give the sequence (5 to 3) of the three stop codons (1 point

each)
________________, ________________, and ________________
5. What is the precise name of the first amino acid put into
bacterial proteins? (must be exact) ________________
6. What is the function of a chaperonin? ________________
7. What is the precise name we give to a gene that after it is
mutated can cause a cancer? ________________
15. What important molecule is an intermediate in the reaction

catalyzed by phosphoglycerate mutase? ________________
16. Name the animal product of fermentation ________________
17. What pathway described in class is an important source of
NADPH? ________________
8. Ras causes uncontrolled growth after it is mutated. What

does the mutation in ras cause it to be unable to catalyze?
________________
9. Name one electron carrier described in class in its oxidized
form ________________
10. Describe how one makes a sugar alcohol ________________
11. Describe the structural dierence between amylose and
amylopectin
12. What animal polysaccharide is similar to amylopectin?
________________
393

1. The initiation of prokaryotic translation is a very intricate
process in which multiple factors must come together to prepare
3. The Cori cycle helps the body to adapt to important situations.

Describe the situation(s) it helps to adapt to. In addition, show
the relevant places in the body where the reactions occur and
show what the products of each relevant organ of the body is. A
diagram will be helpful.
for the elongation phase. Describe all of the factors described in

class, the order in which they come together, and the final
structure at the end of initiation. If you list steps in the elongation
Jump to Chapter
phase, you will lose points.
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
2. A reaction goes as follows

Student <=> Instructor
The reaction goes forward when there are equal amounts of
Student and Instructor. Similarly, the reaction goes forward when
there is ten times as much Student as there is Instructor. Is it
possible for the reaction to go forward when there is ten times as
much Instructor as Student? If yes, you must state why so. If no,
your answer must similarly answer why so. If it is impossible to
answer, your answer must also state why so. Any answer you
give absolutely be based mathematically in energy. Answers
without a mathematical justification will receive no credit.
394
Final Exam (BB 350)

would receive four points if you circled b,c, and d. You would
receive one point if you circled a and b. You would receive no
points if you circled only a. If we have uncertainty about
whether or not an answer is marked, it will automatically be
markings.




1. Hydrogen bonds

A. are weaker than covalent bonds

C. occur as a result of ionization

D. are the bonds formed between cysteines

E. stabilize secondary, tertiary, and quaternary structures

A. glycine is smaller than proline

B. all of them have a pI

C.biological ones are almost always in the D configuration

D. they can be acted upon by transaminases

3. With respect to membrane lipids,

A. sphingolipids contain glycerol

B. glycerophospholipids do not contain fatty acids

C. sphingolipids are common in brain tissue

D. cholesterol is common in brain tissue
4. With respect to mRNAs,

A. they carry the anti-codon

B. there are 64 of them in theory

C. they contain stop codons

D. they have caps in prokaryotes
6. With respect to viruses,

A. HIV is a retrovirus
B. they sometimes carry oncogenesthey can use reverse
C. transcriptase to make RNA from

RNA

D. they can infect prokaryotes and eukaryotes
7. With respect to lipid metabolism

A. fats are more reduced than carbohydrates
B. fatty acid synthesis occurs in the cytoplasm

C. ACP carries fatty acids across the inner mitochondrial

membrane

D. fat breakdown is stimulated by insulin
8. With respect to photosynthesis,

A. protons are pumped outside the chloroplast

B. oxygen is the terminal electron acceptor

C. ATP is produced by substrate level phosphorylation

D. oxygen is produced in the dark cycle
395

9. What is the name of the sequence a few bases before the E.

coli (bacterial) translational start site? ________________
10. What enzyme present in the developing embryo builds the
ends of the linear eukaryotic chromosomes? ________________
1. What does one have to remove in order to convert a weak acid

into a salt? ________________
11. What trait commonly associated with a plasmid allows a

researcher to easily identify which cells get the desired DNA
2. Which amino acid in the catalytic triad of chymotrypsin gains a
during transformation? ________________
proton transiently during the catalytic cycle? ________________

12. What is the name of the enzyme that is essential for removing
3. Explain why myoglobin does not exhibit cooperativity
primers in E. coli DNA replication? ________________
________________________________
13. What protein in translation is responsible for translocation?
4. Define cooperativity ________________
________________
5. How does the pKa of a very weak acid compare to the pKa of a
14. What is on the 5 end of eukaryotic mRNAs that is not on the
weak acid ?
5 end of prokaryotic mRNAs? ________________
________________ (answer must be clear)
15. Draw the Fischer form of D-glucose and its enantiomer (6
6. Explain precisely the piece of information that one would
points)
always know if one had the Km for an enzyme

________________
Glucose = ________________ Enantiomer = ________________
7. Name the strongest force stabilizing tertiary structure
16. When cells are going through fermentation, what molecule
________________
are all of them trying to produce? ________________
8. Which strand in DNA replication contains the Okazaki
17. Define feedback inhibition ________________
fragments? ________________
396
18. Besides glycogen, what other molecule is the substrate of
28. Name an anti-cancer drug discussed in class that mimics
glycogen synthase? ________________
folate ________________
19. What enzyme of gluconeogenesis is inhibited by F2,6BP?

________________
20. Besides acetyl-CoA, what is the only other 2 carbon
molecule in the glyoxylate cycle? ________________
21. What enzyme catalyzes the big bang of glycolysis?
________________
22. Which molecule of the electron transport system is referred to
as the trac cop?

1. One liter of a buer from the blood of Modest Mouse singer
Isaac Brock is found to have a pH of 7.2. The pKa of this buer is
7.4. Does the buer have more salt or more acid? (1 point)
Use the Henderson-Hasselbalch equation to justify your answer.
Your answer MUST be based in the Henderson-Hasselbalch
equation. Other answers will automatically be wrong. (7 points)
________________
23. Name the molecule that directly donates two carbons to the
growing fatty acid chain. ________________
2. Describe the sequence of events involved in the elongation of

translation in prokaryotes. Name the relevant proteins, RNA
24. What enzyme of the cholesterol biosynthesis pathway is
molecules, and structures described in class and the functions
feedback inhibited? ________________
they perform. (9 points)
25. Besides palmitoyl-CoA, what other molecule listed in class is

a precursor of sphingolipids? ________________
26. What molecules are substrates for ribonucleotide reductase
(answer must be precise) ________________
27. When you develop gout, what molecule is precipitating in
your cells? ________________
397
3. A reaction has twice as much product as reactant at

equilibrium. What is the value of the G? ( I am not looking for a
number here) (2 points)
Which direction will the reaction go when there are equal amounts
of reactant and product? (Your answer MUST be based in the G
equation) (6 points)
4. Compare and contrast electron transport and oxidative

phosphorylation with the light reactions of photosynthesis with
respect to electron sources, electron acceptors, movement of
protons, and any products of these reactions.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
398
Exam 1 Key (BB 450/550)
5. You have an acetate buer. You manage to magically destroy

half of the sodium acetate in it without converting it into
anything else. What happens to the pH of the resulting
solution?

points, except as noted. (Answers in RED)
6. Tell precisely/completely what one joins together to make a

peptide bond. Structures not necessary or desired.
1. You have an acetate buer. You add sodium acetate (salt) to it.
What happens to the pH?

The pH increases
2. You are examining a 2D gel. What was the name of the first
Link the carboxyl end of one amino acid to the amino end of
the next one
7. Scurvy arises as a result of the structural weakness of what

compound in the body?
technique that was used to perform this technique?

Isoelectric focusing
3. You want to make the smallest polypeptide you can that will
have the maximum positive charge without using the same
amino acid twice. Give a sequence of such a polypeptide that
you would make

Many possibilities - one is His-Lys-Arg
4. Name all of the amino acids that cannot exist as zwitterions

pH decreases
Collagen
8. Name the categories described in class that the following

amino acids are grouped in
Leucine = Aliphatic
Cysteine = Sulfhydryl
Asparagine = Carboxyamide
There are none
399
9. What chemical named in class can one use to reduce a

covalent bond between two cysteine side chains?

12. What is the name of the technique described in class uses

beads with tiny tunnels in them?
Mercaptoethanol or dithiothreitol
10. A scientist studies the kinetic behavior of an enzyme and

obtains the following plot. Clearly mark the point on the graph
Gel (molecular) exclusion chromatography

13. Name all of the levels/types of protein structure stabilized by
hydrogen bonds
you would use to calculate Km (2 points) and tell precisely how
Secondary, tertiary, quaternary
you would calculate Km from the value of this point (2 points).

1. You have 1L of a 0.6M buer at maximum buering capacity.
When you add 0.1 moles of HCl to the buer (no volume change),
the pH is 9.4. Show how you would calculate the pKa of the
buer.
at maximum buering capacity, [S] = [A]. This means 0.3 moles
of each in 1L. Adding 0.1 moles of HCl converts 0.1 moles of S
into A, resulting in 0.4 moles of S and 0.2 moles of A.
Consequently, the pKa can be calculated as
pKa = 9.4-log{0.4/0.2}

[Product]/time
400
2. A scientist has the polypeptide shown below at pH = 0 and
begins adding NaOH to it.
glycine-arginine-leucine-aspartic acid-methionine-leucine
a. Using the pKa values from the first page of the exam, draw the
titration curve (with properly labeled axes) for this molecule up
to pH = 14.
correct answer.
1. I named at least four molecules/atoms/ions that aect
hemoglobins structure upon binding to it. Name each molecule/
atom/ion and describe precisely where it binds on hemoglobin
and how it aects oxygen anity (20 points)
Protons - binds on amine side chains. Changes hemoglobins
structure to favor release of oxygen
2,3 BPG - binds in the donut hole. Favors T-state - release of
oxygen
Carbon dioxide - binds protonated histidines. Favors release of
oyxgen
Oxygen - binds iron in heme group. Favors R-state - more
binding of oxygen
b. Indicate the charge on the polypeptide at each relevant

place on the graph
c. Show how to calculate the pI of this polypeptide
401
2. Your friend is studying an enzyme in the presence of a noncompetitive inhibitor and says that it gives a value of Kcat dierent
than the same enzyme without the non-competitive inhibitor.
Another friend says that it should have the same Kcat as the
uninhibited reaction. The head of the lab walks up and says that
both statements can be correct, depending on how you calculate
Kcat. What is the head of the lab thinking? (10 points)
In the case of the first friend, he/she is only considering the total
amount of enzyme. The second friend is only considering the
amount of enzyme that is actually active.
Since the same concentration of product is produced in both
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
cases, the ratios

[Product]/[Total Enzyme = active + inactive]
and
[Product]/[Total Enzyme = active]
will be dierent, even though the same concentration of product
exists. Thus, either may be right, depending on the concentraiton
of enzyme used.
402
Exam 2 Key (BB 450/550)
4. Only one of the allosteric eectors of ATCase favors the Tstate. Which one?
CTP

5. Draw the structure of the first glycolysis intermediate

containing two phosphates.

1. What is the dierence between the DNA sequence recognized
by a restriction enzyme and the DNA sequence recognized by
its corresponding methylase?
No dierence
2. Carbonic anhydrase is more active at pH 8.5 than it is at pH 7.
Precisely what is favored at the higher pH that causes this?
Deprotonation of water
3. I talked about at least three dierent ions that could act as
nucleophiles in enzyme catalysis. Draw three of these (hint
6. Precisely what chemical reaction on what molecule/compound/

enzyme does warfarin directly inhibit (four points)?
Carboxylation of glutamate side chains on prothrombin
be sure to draw the form of each ion that acts as the

nucleophile)
7. Define the term anomers

O-
HO- / S-
Two sugar molecules diering in structure

only at the anomeric carbon
403
8. Draw the Fischer structure of and name the only pentose you
are responsible for knowing the structure of (no partial credit)
12. What are the name(s) of the second/third messenger(s) for the
pathway involving phospholipase C?
2nd = IP3
3rd = Ca++
13. Name a compound described in class that is an inhibitor of
BCR-ABL
The sugar is ribose

9. What is the name of the only enzyme of glycolysis that
catalyzes a redox reaction?
JumpGleevec
to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
Glyceraldehyde-3-phosphate dehydrogenase
to move the catalytic triad closer together to create the
alkoxide ion and start the catalytic process?
Binding of the proper substrate at the S1 site
11. Where in the cell are O-linked glycoproteins made?
Golgi apparatus
404
2. The reaction below meets the needs of the bodys muscles
remarkably well. Explain how the G for this reaction varies
according to whether one is exercising or resting and how
(15 points each)
that provides the muscles with what they need to keep the
1. A reaction is at equilibrium at a temperature of 300K with a

[Reactant] = .4M and [Product] = 1.0M. Using as much of
this information as possible, write an equation to calculate
the G when there are equal amounts of the two (15 points)
0 = G + 300RLn[1/.4], so G = -300RLn[1/.4]
then
G = -300RLn[1/.4] + 300RLn[1/1],
but Ln[1/1] = 0, so
G = -300RLn[1/.4]
body functioning. (15 points)

(G = + 12 kJ/mol)
I use C here to mean creatine and CP to mean Creatine
phosphate
1. When exercising, [ATP] falls and [ADP] rises, thus the Ln
term of the G equation,
Ln{[CP][ADP]/[C][ATP]} becomes more positive, moving the
equilibrium to the left, favoring the production of ATP when it
is needed.
2. At rest, the opposite happens, so the Ln term becomes
more negative, favoring storing of energy in the form of CP
405
2. You are walking home one night when out of the bushes jumps
your biochemistry professor singing loudly and very o key. This
scares you terribly, so you start secreting the same hormone you
correct answer.
would if a grizzly bear were chasing you. Name the hormone and
1. You have four friends. Two are born without alpha-1antitrypsin and two are born with a normal alpha-1-antitrypsin.
Their circumstances are below:
A.
B.
C.
D.
No alpha-1-antitrypsin - smokes
No alpha-1-antitrypsin - doesnt smoke
Regular alpha-1-antitrypsin - smokes
Regular alpha-1-antitrypsin - doesnt smoke
show/describe the entire signaling pathway described in class

that it activates. (15 points)
The hormone is epinephrine
The signaling pathway goes as follows
Epinephrine binds to the beta-adrenergic receptor, activating it.
The beta-adrenergic receptor activates a G protein, favoring it
dumping GDP and loading GTP. The activated G protein
Predict the relative tendency to have emphysema you would
activates adenylate cyclase, which makes cAMP. cAMP activates
expect for each of the friends in the comparison below based on
protein kinase A, which in turn favors production of glucose by
class discussion. Justify your answer at the end of the problem.
(ultimately) stimulating enzymes that breakdown glycogen and
(15 points)
activate gluconeogenesis
a. Compare A to B (is emphysema in A > B, A<B, or A=B)

b. Compare C to D (is emphysema in C > D, C<D, or C=D
c. Compare B to D (is emphysema in B > D, B<D, or B=D)
a. A = B
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
b. C > D
c. B > D
406
Final Exam Key (BB 450/550)


[Product]/Time
5. Name three forces stabilizing quaternary interactions

1. One method of separating proteins described in class
combines two dierent separation techniques. Name the
overall method and the names of the two separation
techniques.
hydrogen bonds, ionic bonds, disulfide bonds,

hydrophobic bonds
6. A deficiency of what dietary molecule causes scurvy?
Vitamin C
Overall method = 2D gel electrophoresis.

The two techniques are isoelectric focusing and SDS-PAGE
7. Name the only molecule described in class that competes with

oxygen for the binding site on hemoglobin
Carbon monoxide
2. Name all of the amino acids described in class that can be

phosphorylated
Tyrosine, serine, threonine (histidine can also be phosphorylated,
but it wasnt described in class)
8. To what atom does oxygen bind in hemoglobin?

Fe++
9. Precisely where on ATCase does aspartate bind?
Active site
3. Describe or illustrate (precisely) below the reaction that

mercaptoethanol stimulates
Mercaptoethanol reduces disulfide bonds to sulfhydryl bonds
407
10. What is the name of the enzyme in glycolysis that produces

an intermediate that aects hemoglobins anity for oxygen?
Phosphoglycerate mutase
11. Name the only enzyme in glycolysis that is regulated both
allosterically and by covalent modification
Pyruvate kinase
12. Draw the Haworth structure for the only hexose given in class
for which you must know the furanose form
15. Gleevec inhibits Bcr-Abl. What enzymatic reaction does this

protein catalyze?
Phosphorylation of tyrosine
16. What bond is broken by the nucleophilic attack in the fast
step of the catalytic mechanism of serine proteases?
Peptide bond
17. Name three first messengers described in class
ephinephrine, glucagon, insulin
18. A friend tells you that a new buer has been discovered in
which there is more salt than acid when the pH is the same as
the pKa. Using math, explain why this is true or false. (Your
answer must also say TRUE or FALSE)
13. What is the name of the enzyme that catalyzes the

breakdown of blood clots?
Plasmin
to move the catalytic triad closer together to create the
alkoxide ion and start the catalytic process?
False
pH = pKa + Log {[Salt]/[Acid]}.
Thus if pH = pKa, then Log {[Salt]/[Acid]} must be zero, but the
ratio of [Salt]/[Acid] is not equal to one, so the statement must be
wrong.
Proper substrate must bind at the S1 site
408
19. Kevin Aherns hypothesis of why Americans are becoming
24. Name the only enzyme I described in class that is used both
more and more obese says it is due to the bypassing of an
in the breakdown of glycogen pathway and the synthesis of
important enzymatic reaction. What is the name of this
glycogen pathway
enzyme?
Phosphoglucomutase
Phosphofructokinase (PFK)
20. What molecule is synthesized in animals in order to make

NAD+ when oxygen levels are low?
25. What does it take to convert glycogen phosphorylase a from

the T state to the R state?
Nothing. It automatically flips
Lactate
21. In utilization of galactose, it is first phosphorylated and added
to a molecule that results in the release of glucose-1-
Jump to Chapter
phosphate. To what molecule is the phosphorylated

galactose attached?
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
UDP
22. Name a transcription factor made/activated in cells when

oxygen is lacking.
HIF-1
23. Name a molecule that activates an enzyme in feed-forward
activation.
F1,6BP (Fructose-1,6-bisphosphate)
409
2. A reaction starts with 5 times as much reactant as product,
but at equilibrium, it only has 3 times as much reactant as
product.
points each)
a. Write an equation to show how to calculate G (3 points)
1. You have 1L of a 0.6M buer with twice as much salt as acid.

After you add .1 moles of HCl (no volume change), the pH is
8.32. Write an equation to calculate the pH before the HCl was
0 = G + RTLn[1/3].
G = -RTLn[1/3]
b. Write an equation to show how to calculate G at the very
beginning (3 points)
added.
To start, you have 0.4 moles salt and 0.2 moles acid
G = -RTLn[1/3] + RTLn[1/5]
Upon adding the HCL, you have 0.3 moles of salt and acid
c. Show how you would determine the sign of the G (3 points)
Thus, the pH at this point is the pKa, as well.
Since 1/3 <1, the log term must be negative,
Consequently, before the HCl was added, the pH is given as

follows:
pH = 8.32 + Log(0.4/0.2)
so the G must be positive

d. What was the sign of G at the start of the reaction? Why? (3
points)
Since the reaction goes from 5 times as much reactant as
product to only 3 times as much reactant as product, the reaction
must have run forwards. Thus, the G was negative then.
410
3. An analysis of an enzymatic reaction is performed in the
c. What type of inhibition is occurring? (2 points)
presence and absence of an inhibitor. The inhibitor appears only
Non-competitive inhibition
to reduce the Vmax of the enzyme.

a. Draw a single Lineweaver Burk plot showing the uninhibited
and the inhibited reaction. Clearly label all relevant features on
the graph. (5 points)

correct answer.
1. Regulation of glycolysis and gluconeogenesis is complex. It
involves allosteric interactions, covalent modifications, and
control of enzymes that make/break down molecules that aect
the pathway.
a. Name the enzymes regulated in each pathway (7 points)
b. Draw a single V vs. [S] plot for the showing the uninhibited and
the inhibited reaction. Clearly label all relevant features on the
graph. (5
points)
Glycolysis - hexokinase, PFK, pyruvate kinase

Gluconeogenesis - pyruvate carboxylase, PEPCK, F1,6BPase,
G6Pase
b. Name one allosteric activator and one allosteric inhibitor of the
reciprocally regulated enzmes above(4 points)
PFK - F2,6BP, AMP activators. ATP, citrate inhibitors
F1,6BPase - Citrate activator, F2,6BP, AMP inhibitors
411
c. Name the most important allosteric regulator of the two
3. We have talked this term about three broad ways in which
pathways and show precisely how its synthesis and breakdown is
enzyme activity can be regulated. Name each way. Name one
regulated. (6 points)
enzyme regulated each way. Give detail as appropriate for each
Most important regulator is F2,6BP.
means of regulation. If external molecules or modifications are

relevant, your answer must include them and a description must
Its synthesis is activated by dephosphorylation of FBPase2/PFK2
include the eect the molecules or modifications have on each
by phosphoprotein phosphatase (ultimately activated by insulin).
enzyme. (12 points)
Its breakdown is activated by phosphorylation of FBPase2/PFK2
A. Allosteric regulation - several examples. One is ATCase,
by Protein Kinase A (ultimately activated by epinephrine)
which is allosterically activated by ATP and allosterically

inhibited by CTP
2. Glycogen phosphorylase is regulated allosterically and by an

enzyme it carries around with it. Draw a scheme illustrating all of
B. Covalent modification - several examples. One is glycogen
the ways in which glycogen phosphorylase is regulated and also
synthase, which is inactivated by phosphorylation by protein
a scheme showing how the enzyme it carries can aect the
kinase A and activated by dephosphorylation by
enzyme. (10 points total)
phosphoprotein phosphatase.
C. Control of whether an enzyme is made or not. A good
example is PEPCK in gluconeogenesis. It is not made in many
tissues, but is made in liver and kidney cells where
gluconeogenesis is active
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
412
Exam 1 Key (BB 451/551)

This is basically a True/False type of questioning. True answers
below are marked in RED

A.
B.
C.
D.
has students from all over the world.

A. succinate dehydrogenase requires FAD for catalysis
B. it includes two decarboxylations not found in the

glyoxylate cycle

C. NADH is not used

D. it uses oxygen in one or more of its enzymatic reactions
2. With respect to respiratory control,

A. cyanide will stop oxidative phosphorylation first

B. exercising will favor electron transport

C. resting will favor decrease in ADP

D. 2,4 DNP (miracle diet drug) will increase NADH and

temperature
3. With respect to membrane transport,

A. lactose permease is an anti-port

B. it requires ATP

C. it requires diusion through the membrane

D. it cannot be electroneutral if sodium is involved
4. With respect to electron transport,

A. electrons get pumped into the intermembrane space

B. oxygen is the terminal proton acceptor

C. the Q cycle occurs in Complex III

D. 2,4 DNP will increase oxygen consumption

A. increasing oxygen will increase the proton gradient

B. lack of NAD+ will ultimately reduce electron flow

C. uncoupling protein (UCP) will reduce ATP production

D. the citric acid cycle will run fastest when electron

transport runs fastest
413
Section II: (50 points total) Each sentence below in this

section is missing a word or phrase to complete it. Fill in the
blank as appropriate to complete the sentence with a correct
statement. Each correctly filled in blank below will be
awarded two points.
1. Name the five carbon intermediate in the citric acid cycle
alpha-ketoglutarate
2. Which enzyme of the citric acid cycle catalyzes a reaction pulls
a reaction preceding it?
citrate synthase
3. Which enzyme of the citric acid cycle requires lipoamide?
alpha-ketoglutarate dehydrogenase
4. Which enzyme of the citric acid cycle catalyzes the only
oxidation that doesnt produce NADH?
succinate dehydrogenase
5. What citric acid cycle intermediate can be readily made from
glutamic acid in an anaplerotic reaction?
alpha-ketoglutarate
6. How many oxaloacetates are produced per turn of the citric
acid cycle?
one
7. Name a poison described in class that reacts with a citric acid

cycle coenzyme
arsenic
8. Which class of membrane-related proteins would be the most
likely to have hydrophobic amino acids on its exterior?
integral
9. If one membrane protein pump were missing from the cell,
neural transmission as described in class would not be
possible. What pump is that?
Na+/K+ ATPase
10. What is the basis for exclusion of potassium from a sodium
channel?
size
11. What is the basis for exclusion of sodium from a potassium
channel?
energy of hydration/dehydration
12. Active transport always has at least one mechanistic
dierence from passive transport. It is the reason that active
transport requires energy. What is that dierence?
In active transport, at least one molecule is being moved against
a concentration gradient
414
13. What is the driving force for passive transport?
19. A mitochondrion is treated with 2,4 dinitrophenol (2,4 DNP).

What happens to its oxygen consumption?
diusion
increases
14. What is the definition of a symport?
a membrane transport system that moves two molecules in the
same direction across the membrane
20. The mitochondria in a cell are tightly coupled. The citric acid
cycle is running furiously. What normal explanation (not exotic
or unusual) makes the most sense about what this cell is
doing to cause this?
15. You remove digitoxigenin from a heart cell that had been
treated with it. What happens to the concentration of sodium
outside of the cell?
Cell is burning ATP rapidly
increases
16. In the Q cycle, what takes electrons away from Complex III?
cytochrome C
17. Complex V has a rotating complex that make ATP. It has
three dierent configurations. What is the form of the
configuration where ATP is released?
21. If one wanted to make a photosynthetic fish, as described in

class, what protein would you need to do it?
bacteriorhodopsin
22. Describe the numbering system for an omega fatty acid.
Which carbon is number one?
methyl carbon
Open (O)
23. What is the name of the category of ATP-using transport
proteins that involve a phosphoaspartate?
18. You treat a mitochondrion with 2,4 DNP and cyanide. What
happens to oxygen consumption?
Little or nothing
P-type ATPases
24. What is the charge on a copper atom in Complex IV after it
has accepted an electron?
+1
415
25. If you wanted to raise the Tm of a membrane, how would you

alter the chemical composition of its fatty acids?
make them longer and/or more saturated
Section III: (30 points total) Matching.

T
M
U
R
L
G
D
A
C
B
C
E

I
Q
V

1. Na/K ATPase

A. Can stop oxygen consumption indirectly
2. Isocitrate

B. Limiting for people not exercising
3. Brown fat

C. Produced by superoxide dismutase
4. Acetaldehyde

D. Glyoxylate cycle enzyme requirement
5. Decarboxylation

E. Carries electrons in pairs
6. Sphingomyelin

F. Five carbon intermediate
7. Malate synthase

G. Phosphorylated membrane component
8. Antimycin A

H. Neurotransmitter described in class
9. Oxygen

I. Neurotransmitter holder
10. ADP

J. Complex IV inhibitor
11. H2O2

K. Citrate synthase byproduct
12. Coenzyme Q

L. Process that does not occur in glyoxylate

cycle
13. Synaptic vesicle

M. Six carbon intermediate
14. Malate

N. Symport
15. Thiamine pyrophosphate (TPP) O. Blocked by tetrodotoxin

P. Produced by citrate synthase

Q. Gets oxidized after being shuttled

R. Ethanol precursor

S. Produced by oxidative phoshorylation

T. Electro-genic
U. Can become uncoupled
V. Carries a two carbon group
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
416
Exam 2 Key (BB 451/551)

These are basically True/False question. True answers are show
in RED



D. has students from all over the world
1. With respect to cholesterol metabolism,

A. it uses palmitoyl-CoA and serine

B. it includes an intermediate of squalene

C. ATP is not used
D. it is a precursor to vitamin D, bile acids, and

steroid hormones
2. With respect to fatty acid metabolism,

A. it starts with oxidation of medium chain fatty acids in

peroxisomes
B. oxidation of saturated fatty acids involves cis

intermediates

C. enoyl-CoA isomerase is not needed for oxidation of

saturated fatty acids
D. ketone bodies use the last enzyme of fatty acid

synthesis

A. ibuprofen is a COX-1 inhibitor

B. steroids inhibit COX-2 enzymes
C. they are usually produced from thromboxanes or

leukotrienes

D. PLA-2 inhibits the action of aspirin
A. deoxyribonucleotides are made from

B. PRPP amidotransferase balances relative amounts of

pyrimidines

C. NDP Kinase (NDPK) converts ADP to dADP

D. dUTPase converts dUTP to dTTP
5. With respect to DNA replication,
A. the beta clamp is responsible for making DNA

Polymerase III more processive

B. helicase forms double helices at the rate of 6000 rpm
C. Okazaki fragments are only made in the 5 to 3

direction

D. lagging strand replication occurs in the 3 to 5 direction
417
Section II: (50 points total) Each sentence below in this section is
missing a word or phrase to complete it. Fill in the blank as
Each correctly filled in blank below, except as noted, will be
awarded two points.
6. What is the name of the last product of beta oxidation of a

fatty acid with an odd number of carbons?
propionyl-CoA
7. Name the only regulated enzyme in either fat or fatty acid
breakdown
1. What is the name of the transcription factor that helps control

synthesis of HMG-CoA reductase?
SREBP
triacylglycerol lipase
8. Draw the structure that the enzyme thiolase would cut
2. I named four general mechanisms for regulating HMG-CoA

reductase. Name three of them (1 point each)
control of synthesis, allosterism, covalent modification,
half-life (time to destruction)
3. What group of molecules has their synthesis stopped by
aromatase inhibitors?
9. Describe accurately in words what enoyl-CoA

isomerase catalyzes
estrogens
4. I mentioned three factors to consider for controlling the level of
cholesterol in the body. Name them (1 point each)
diet, synthesis, storage/recycling
5. What is the name of the cellular structure missing in familial
hypercholesterolemia?
liver receptor for LDLs
Conversion of cis bonds in fatty acids between carbons 3-4 into

trans bonds between carbons 2-3
10. Name the enzyme that catalyzes the first reaction in the
pathway leading to ketone bodies
thiolase
11. Name the molecule produced by catalysis by the only

regulated enzyme of fatty acid biosynthesis
418
malonyl-CoA
12. Name the molecule that shuttles the precursor of fatty acid
synthesis to the cytoplasm
citrate
13. How does carbamoyl phosphate synthetase protect its
intermediate product from water?
by moving it through a tunnel
14. What is the name of the enzyme that balances purines and
pyrimidines in de novo ribonucleotide synthesis?
Oxygen
19. What is the name of the electron source for fatty acid
biosynthesis?
NADPH
20. What is the name of the electron acceptor in the first step of
fatty acid oxidation?
FAD
21. Name the form of DNA discovered by Rosalind Franklin
A-form
ATCase
15. What molecule is the energy source for de novo synthesis of
AMP?
22. Name the enzyme necessary for joining the DNAs of Okazaki
fragments
DNA ligase
GTP
16. What is the name of the molecule that is the branch point in
de novo purine ribonucleotide synthesis?
22. What is the name of the first protein to bind to the E. coli DNA
replication origin?
DNA-A
Inosinate (IMP)
17. Write out the reaction catalyzed by adenylate kinase
AMP + ATP <=> 2ADP
23. Precisely what does telomerase use as a template to make

telomeres?
an RNA that it carries
18. What atom does RNR (ribonucleotide reductase) catalyze the

removal of?
419
24. What enzymatic activity is present in DNA polymerase I and

III, but lacking in reverse transcriptase of HIV?
Proofreading (3 to 5 exonuclease activity)
25. Give the ratio that determines whether or not a DNA is
relaxed or not (note I am looking for the units, not a number)
bp/turn
Matching
U 1. dnaB

M 2. Guanosine

L 3. Gyrase

I 4. Arachidonic acid

B 5. LDL

P 6. Serine

N 7. CDP

F 8. Cholesterol

V 9. dATP

R 10. S-Adenosyl Methionine (SAM)
S 11. HMG-CoA

A 12. Lipase

J 13. Ganglioside

E 14. ACP

T 15. Glycerol

A. Necessary for fat digestion

B. Lipoprotein complex
C. Palmitate precursor
D. Requires vitamin B12
E. Replaces CoA of malonyl-CoA
F. Feedback inhibitor of long pathway
G. ATCase inhibitor
H. Lacking in Lesch-Nyhan syndrome
I. Leukotriene precursor
J. Sphingolipid
K. Prostaglandin product
L. Topoisomerase
M. Nucleoside
N. Found in activated intermediates
O. Covalent modification product
P. Necessary for sphingolipid

synthesis
Q. Glycerophospholipid
R. Methyl donor
S. INSIG target
T. Can be made into glucose
U. Helicase
V. Allosteric inhibitor of

ribonucleotide reductase
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
420
Final Exam Key (BB 451/551)

sentence to make a correct statement. Points will be awarded
for each circled response that makes a correct statement and for
These are basically True/False questions. True answers are
marked below in RED





A. GDP is required

B. lipoic acid has no role

C. oxygen is not a substrate in the reactions of the cycle

D. oxidative decarboxylation does not occur
2. With respect to movement of compounds across biological

membranes,

A. active transport requires ATP

B. water cannot readily cross membranes

C. a proton gradient is required

D. protons generally require a protein to move across

membranes

A. oxidative phosphorylation will speed up to make up for

an oxygen deficit

B. low ADP levels will have the same overall eect as

low oxygen levels

C. increasing intermembrane space pH will reduce ATP

production

D. cyanide will reduce both ATP production and the

proton gradient
4. With respect to the Q cycle,

A. it explains how electrons arrive in pairs, but are

converted to moving singly

B. it involves coenzyme Q in three dierent forms

C. Cytochrome C carries electrons to complex III

D. each QH2 sends electrons to two dierent things
5. With respect to fatty acid oxidation,

A. it occurs in the cytoplasm and the peroxisomes

B. it produces FADH2 and NADH

C. it only occurs on fatty acids shorter than 16 carbons
D. it occurs when the fatty acid is bound to ACP
421
6. With respect to fatty acid synthesis,

A. it is allosterically activated by palmitate/palmitoyl-CoA

B. it occurs in the cytoplasm and endoplasmic reticulum

C. it uses malonyl-ACP
D. it requires a carboxylation and a decarboxylation

A. aspirin is a COX inhibitor

B. inhibiting phospholipase A2 will stop release of

fatty acids from membranes

C. they are precursors of cholesterol

D. they are pain relievers
Section II: (80 points total) Each sentence below in this section
is missing a word or phrase to complete it. Fill in the blank as
Each correctly filled in blank below will be awarded two points.
1. What coenzyme named in class does arsenate react with?
lipoamide (lipoic acid)

A. it uses ribonucleotide reductase to convert all dNDPs to

dNTPs

B. it uses CTP to make UTP

C. it uses dTMP to make dUMP

D. it uses DHFR to make dNDPs from NDPs
9. With respect to transcription,

A. it occurs at a structure called a replication fork
B. the coding strand is complementary to the RNA being

made

C. it makes RNA in the 3 to 5 direction

D. it requires a ribosome
10. With respect to gene expression,

A. attenuation involves early transcriptional termination

B. the lac repressor binds to DNA when it binds allo-lactose

C. CAP binds to DNA when it binds to cAMP
D. IRE-BP binds to DNA when it does not bind iron
2. Which enzyme of the citric acid cycle catalyzes a reaction that produces FADH2?
succinate dehydrogenase
3. What is the definition of an antiport?
a membrane transport protein that moves two molecules in
opposite directions across the membrase
4. Which enzyme of the citric acid cycle catalyzes a reaction that pulls the one preceding it?
citrate synthase
5. What amino acid is alpha-keto-glutarate readily converted to in an anaplerotic reaction?
glutamic acid
6. Name one enzyme unique to the glyoxylate cycle
malate synthase or isocitrate lyase
422
7. You take a heart cell that was treated with digitoxigenin
13. A cell (and its mitochondria) are treated with 2,4 dinitro-
and remove the digitoxigenin. What happens to the concen-
phenol (2,4 DNP). What happens to the citric acid cycle
tration of calcium outside of the cell?
in this cell?
increases
8. Name a specific sphingolipid named in class that contains a phosphate
speeds up
14. Name one of the two isoprenes described in class
isopentenyl pyrophosphate or dimethylallyl pyrophosphate
sphingomyelin
15. Complex V has a rotating complex that makes ATP. It
9. Name the class of lipid described in class that contains
complex carbohydrates
has three dierent configurations. What is the form of

the configuration where ATP is released?
gangliosides
10. A gradient of which ion is used as an energy source to
remove calcium ions from heart cells?
the O configuration
16. Describe precisely the catalytic activity of DNA Polymerase I that removes RNA primers.
sodium
11. What is the criterion we use to determine if a transport
mechanism is active versus passive?
5 to 3 exonuclease
17. Precisely what does telomerase copy to make a telomere?
active transport always has at least one molecule/ion moved
a strand of RNA it carries
against a concentration gradient

18. How does one completely inhibit PRPP amidotransfe12. In what class of membrane transporters does phosphoaspartate appear?
rase?
with high amounts of both AMP and GMP
P-type ATPases
423
19. What is the name of the molecules that donate single car-
25. Name a covalent modification of an enzyme in fatty acid
bons in nucleotide metabolism (hint Im not looking for
metabolism and the eect of that covalent modification
carbon dioxide or bicarbonate) ?
Enzyme modified = acetyl-CoA carboxylase
folates
Modification = phosphorylation / dephosphorylation

Eect of modification = reduce activity / increase activity
20. What molecule does aspirin prevent from being acted

on by COX?
26. Name the enzyme used in DNA footprinting

arachidonic acid
21. In fatty acid synthesis, describe the chemical step after

deyhdration (all I need is a name describing the chemical
DNAse
27. Name the specific catalytic entity that forms phosphodiester bonds between ribonucleotides.
reaction)
RNA Polymerase
hydrogenation
28. Name the specific catalytic entity that forms peptide
22. Name the enzyme that catalyzes conversion of fatty ac-
bonds between amino acids in bacteria
ids with a cis double bond between carbons 3/4 to a
23S rRNA
trans double bond between carbons 2/3?

enoyl-CoA isomerase
29. Define the term template strand

the strand of DNA that is read by RNA Polymerase during
23. What causes gout?
transcription. It is complementary to the RNA that is made
overproduction of uric acid arising from purine catabolism

30. Name the protein described in class that unwinds DNA
24. Where does one find acyl-CoA dehydrogenases that act
on fatty acids of 20 carbons?
at 6000 rpm
helicase
peroxisomes
424
31. In attenuation of the trp operon, what is the function of

the structure in the mRNA that only forms when there is
abundant tryptophan in the cell?
termination of transcription
32. What does tamoxifen prevent the estrogen receptor
from interacting with?
co-activators
33. In elongation, which direction is the ribosome moving
relative to the mRNA?
5 to 3
34. Name the 7TM of vision that is bound to Vitamin A
rhodopsin
35. Name the G protein involved in the sense of smell
Golf
36. Describe the unusual bond in a lariat structure
5 to 2
37. Describe what rho does and how it does it
termination of transcription - climbs the RNA and lifts
the RNA Polymerase o of the DNA it is copying
38. Precisely where is the wobble base of the anticodon
found ?
5 end of the anticodon
Section III: Matching. Each term on the left has a phrase or term on the
right which best describes or matches it. Place the letter of the term/
phrase on the right in the blank before the term on the left that it best
matches. Only one letter is appropriate in the blank. Note that there are
more terms on the right than there are blanks, so not every term on the
right has a best match. Terms on the right may be used once, more than
once, or not at all. If we cannot read your writing or if you put two letters in
any blank on the left, your answer will be counted wrong automatically.
Each correctly matched pair is worth two points.
M 1. TPP

N 2. Intron
I 3. Transducin
P 4. Gyrase
U 5. dATP
H 6. Inducer
J 7. Thiolase

V 8. Statin

L 9. Active transporter
X 10. Tamoxifen

D 11. Trans fat

G 12. Okazaki fragments
T 13. Carnitine

E 14. Anti-codon
A 15. Ribozyme

A. Peptide bond catalyst(s)

B. Purine breakdown product(s)
C. Binds retinal
D. Hydrogenation byproduct(s)
E. tRNA component(s)
F. Spingolipid(s)
G. Lagging strand synthesis product(s)

H. IPTG
I. G protein(s)
J. Ketone body enzyme(s)
K. Prostaglandin enzyme(s)
L. Na/K ATPase
M. Pyruvate dehydrogenase complex

component(s)
N. pre-mRNA component(s)
O. Reverse transcriptase(s)
P. Topoisomerase(s)
Q. Helicase(s)
R. Translation chain terminator(s)
S. Copper
T. Mitochondrial membrane crosser(s)
U. Ribonucleotide reductase inhibitor(s)
V. HMG-CoA reductase inhibitor
W. Fatty acid synthase end product(s)
X. Estrogen receptor binder(s)
425
Exam 1 Key (BB 350)

markings. These are essentially true/fall questions. True answers
are shown in RED.




1. With respect to hydrogen bonds,

A. they are weaker than covalent bonds

B. they are weaker than disulfide bonds

C. they are what make peptide bonds
D. water does not have to be involved

A. glycines R group is a hydrogen

B. lysines R group is more hydrophobic than leucines

C. methionine is the only one with a sulfur in the R group

D. every one of them can exist as a zwitterion
3. With respect to enzymes,

A. Km is a velocity

B. Vmax/2 is the same as Km

C. Kcat varies with substrate concentration

D. Kcat = Km /[enzyme]
4. With respect to information in the syllabus,

A. there is no fixed grading scale

B. grades are curved

C. the grading scale is 90/80/70/60

1. Describe the dierence between a strong acid and a weak acid

in solution.
A strong acid completely dissociates in an aqueous solution,
whereas a weak acid does not
2. Name an amino acid that makes disulfide bonds
Cysteine
426
3. How does a fibrous protein dier from a globular protein?

Fibrous proteins have little tertiary structure, whereas globular
proteins have much tertiary structure
4. A solution has a pH of 6. Write an equation for its hydroxide
ion concentration
9. What units does KM have?

concentration (molarity, for example)
10. Name three separation techniques described in class
gel exclusion chromatogrphy, gel electrophoresis, PAGE, ion
exchange chromatography, anity chromatography
[OH-] = 10-8M
5. What is the name of the group in myoglobin that contains the
iron?
11. What name do we give to the molecule(s) that an enzyme

catalyzes a reaction on?
substrate(s)
heme
6. Define allosterism
when binding of a small molecule to an enzyme aects the
enzymes activity
7. You have a buer solution with more salt than acid and the pH
is 6. What can you say about the pKa?
the pKa is less than 6
8. Explain from an energetic perspective how enzyme catalysis
works (one sentence should suce).
Enzymes work by lowering the activation energy of the reactions
they catalyze
427
b. Calculate the approximate charge this amino acid would have
at a pH of 5.5 (4 points)
1. Explain at the molecular level why smokers breathe more
Zero
heavily when exercising than non-smokers. (16 points)
3. For the following problem, ORGANIZE and LABEL your
Smokers have a higher concentration of 2,3 BPG in their

bloodstream than non-smokers. 2,3 BPG binds to hemoglobin
calculations clearly. No partial credit will be given without clearly
and favors the T state, thus reducing how much oxygen it can
labeled calculations. You do NOT have to enter the numbers into
carry. As a result, smokers blood can carry less oxygen than
a calculator. You simply need to show the last step before

punching it in.
non-smokers blood.
2. An exotic amino acid is discovered that has two amine groups
You have a 1L of a 0.6M buer of ahernium (pKa = 9.11) in which
and three carboxyl groups. The pKa values of the groups are
the concentration of acid is twice that of the salt.
below
a. Write an equation to calculate the pH of this buer (4 points)
Amino #1 = 8.8
Amino #2 = 12.1
Carboxyl #1 = 1.1
Carboxyl #2 = 4.2
Carboxyl #3 = 6.6
pH = 9.11 + log{[.2]/[.4]}
b. If you wish to get the buer to maximum buering capacity,
describe whether you would use HCl or NaOH and how many
moles you would use. You will need to clearly show and label
a. Draw and
your calculations to explain your answer. (16 points)
label
CLEARLY a
You will need 0.1 moles of NaOH. After adding this, you will have
titration curve
0.3M salt and 0.3M acid
with all
Jump to Chapter
relevant
values for this
amino acid
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
(12 points)
428
Exam 2 Key (BB 350)


are shown in RED.




1. With respect to fatty acids,

A. more unsauration lowers their melting temperatures

B. greater length raises their melting temperatures
C. oils have more unsaturated fatty acids than fats

D. fish membranes have more unsaturated fatty acids

than humans
2. With respect to components found in nucleic acids,

A. adenine does not contain a sugar

B. nucleosides do not contain a sugar

C. nucleotides do not contain a phosphate

D. RNA contains ribose, but not deoxyribose
3. With respect to membranes,
A. integral membrane proteins project through both

sides of the lipid bilayer

B. liposomes are human made structures

C. carbon dioxide cannot cross the lipid bilayer

D. cholesterol raises the transition temperature of a

membrane
4. With respect to control of enzyme activity,

A. covalent modification always activates enzymes

B. allosterism always inactivates enzymes

C. feedback inhibition works allosterically

1. What molecule found in the membranes of cells widens the

cells transition temperatures (Tm values)?
cholesterol
429
2. Define allosterism (no points for being close)

when binding of a small molecule to an enzyme aects the
enzymes activity
3. ATCase has two dierent kinds of subunits. What category of
8. Name an inhibitor of prostaglandin synthesis

aspirin, ibuprofen (other COX inhibitors)
9. What chemical change happens to vitamin A that results in
nerve signaling?
subunit does aspartate bind to?

catalytic subunits
4. Carbohydrates are only found attached to the outside of cell
membranes. What is one function, as described in class?
cellular identity
5. What is the driving force for the movement of molecules in
facilitated diusion?
cis-trans bond isomerization

10. What is the chemical dierence between a
deoxyribonucleotide and a deoxyribonucleoside (be precise in
your answer)?
a deoxyribonucleoside has no phosphate, whereas a
deoxyribonucleotide has at least one phosphate
11. In attenuation of the trp operon, when there is sucient
concentration dierence (diusion)

6. What three carbon molecule forms the backbone of
tryptophan, what process is terminated?

transcription
glycerophospholipids, but not sphingolipids?

glycerol
7. What is required for a membrane transport system to be
described as active transport?
at least one molecule must be moved against a
concentration gradient
430
4. The lac operon has controls that allow it to adapt to the cells
needs for energy. Explain all of the controls listed in class and
1. E. coli DNA polymerase has three essential activities for DNA
how they allow for this adaptation.
replication. Name and explain the enzymatic activity that each

lac repressor - binds to lac operator when lactose absent, turning
one catalyzes.
o transcription of the operon. When lactose present, it binds to

5 to 3 DNA polymerase - makes DNA
3 to 5 exonuclease - proofreading - removes mispaired bases
5 to 3 exonuclease - removes RNA primers
2. Draw out the central dogma and identify the exception to it
with respect to retroviruses, such as HIV
DNA -> RNA -> Protein
The exception of retroviruses is that they can make DNA using
RNA as a template
3. I described in class how E. coli can switch from making one
the lac repressor and prevents it from binding to the lac operator,
thus allowing transcription to occur
CAP - when bound to cAMP it binds near the lac operator and
stimulates transcription to occur
5. List and describe three modifications to eukaryotic mRNAs that
do not happen to prokaryotic mRNAs.
5 cap
3 poly-A tail
splicing
class of genes to making another class of genes, due to

changes of circumstances. Describe precisely what is involved
in making this switch (hint this has nothing to do with
lactose).
switching of classes of genes in E. coli can happen as a result of
changing which sigma factor is being synthesized
431
Exam 3 Key (BB 350)

are shown in RED.




1. With respect to tools for biotechnology,
A. a plasmid is a circular DNA that replicates in human cells
B. a polylinker is a short stretch of DNA with multiple
restriction sites in a plasmid

C. enzymes used to cut DNAs at specific places are called

methylases
D. a plasmid for making protein in a cell would have to

have a promoter
2. With respect to sugar structures,

A. a hexose cannot be a furanose
B. the Fischer straight chain structure of glucose has no

anomeric carbon
C. a reducing sugar is easily reduced
D. a glycoside always has a bond involving the

anomeric carbon
3. With respect to translation,
A. the 23s rRNA catalyzes the formation of peptide

bonds
B. initiation does not involve any peptide bond

formation

C. EF-Tu is involved in translocation

D. ATP is required for translocation
4. With respect to polysaccharides,

A. cellulose is a polymer of glucose
B. chitin is a polymer of fructose
C. bacterial cell walls contain peptidoglycan structures

with D-amino acids

Each correctly filled in blank below will be awarded three points,
except as noted. You MUST be precise in your definitions.
1. What is the name of the bacterial protein factor responsible for
translocation during translation?
EF-G
432
2. Precisely where is the wobble base of the anti-codon?

5 end of anti-codon
8. Ras causes uncontrolled growth after it is mutated. What does

the mutation in ras cause it to be unable to catalyze?
conversion of GTP to GDP
3. Describe the physical appearance of a messenger RNA during

eukaryotic translation
9. Name one electron carrier described in class in its oxidized

circularized
form
NAD+, FAD, or NADP+
4. Give the sequence (5 to 3) of the three stop codons (1 point

each)
10. Describe how one makes a sugar alcohol

UAA, UAG, and UGA
5. What is the precise name of the first amino acid put into
bacterial proteins? (must be exact)
f-met (formyl-methionine)
6. What is the function of a chaperonin?
assist proteins in folding properly
7. What is the precise name we give to a gene that after it is
reduce a sugar
11. Describe the structural dierence between amylose and
amylopectin
amylose is a polymer of glucose with only alpha-1-4 linkages
amylopectin is a polymer of glucose with alpha-1-4 linkages plus
alpha 1-6 linkages every 30-50 residues
12. What animal polysaccharide is similar to amylopectin?
glycogen
mutated can cause a cancer?

proto-oncogene
13. Draw the structure of L-glucose in the

Fischer form
433
14. Draw the structure of alpha-D-ribose as a furanose

1. The initiation of prokaryotic translation is a very intricate
process in which multiple factors must come together to
prepare for the elongation phase. Describe all of the factors
described in class, the order in which they come together, and
the final structure at the end of initiation. If you list steps in the
elongation phase, you will lose points.
15. What important molecule is an intermediate in the reaction

catalyzed by phosphoglycerate mutase?
2,3-BPG
16. Name the animal product of fermentation
Elongation involves three initiation factors, IF-1, IF-2, and IF-3.

These combine first with the 30S ribosomal subunit and the
mRNA where the Shine-Dalgarno site of the mRNA is lined up
with a complementary region of the 16S rRNA. Then the initiator
tRNA (f-MET) and large subunit are brought to the system to
create the intact ribosome with mRNA running through it and a
single tRNA-fMET in the P-site.
lactate
17. What pathway described in class is an important source of
NADPH?
pentose phosphate pathway
434
2. A reaction goes as follows

Student <=> Instructor
The reaction goes forward when there are equal amounts of
Student and Instructor. Similarly, the reaction goes forward when
there is ten times as much Student as there is Instructor. Is it
possible for the reaction to go forward when there is ten times as
much Instructor as Student? If yes, you must state why so. If no,
your answer must similarly answer why so. If it is impossible to
answer, your answer must also state why so. Any answer you
give absolutely be based mathematically in energy. Answers
without a mathematical justification will receive no credit.
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
Yes, it is possible. If the G value is suciently negative, it can

counterbalance any positive values arising from the scenarios
described above
3. The Cori cycle helps the body to adapt to important situations.

Describe the situation(s) it helps to adapt to. In addition, show
the relevant places in the body where the reactions occur and
show what the products of each relevant organ of the body is. A
diagram will be helpful.
Liver - absorbs lactate from bloodstream and converts it to
glucose. Glucose exported to bloodstream
Muscles - absorb glucose from bloodstream and produce lactate,
which they export to the bloodstream.
The process occurs during periods of exercise when the body is
unable to provide sucient oxygen to the muscles.
435
Final Exam Key (BB 350)

would receive four points if you circled b,c, and d. You would
receive one point if you circled a and b. You would receive no
points if you circled only a. If we have uncertainty about
whether or not an answer is marked, it will automatically be
are shown in RED.

A. glycine is smaller than proline

B. all of them have a pI

C.biological ones are almost always in the D configuration

D. they can be substrates for transaminases

3. With respect to membrane lipids,

A. sphingolipids contain glycerol

B. glycerophospholipids do not contain fatty acids

C. sphingolipids are common in brain tissue

D. cholesterol is common in brain tissue
4. With respect to mRNAs,

A. they carry the anti-codon

B. there are 64 of them, in theory

C. they contain start and stop codons

D. they have caps in prokaryotes




6. With respect to viruses,

A. HIV is a retrovirus

B. they sometimes carry oncogenes

C. they can use reverse transcriptase to make RNA from

DNA

D. they can infect prokaryotes and eukaryotes
1. Hydrogen bonds

A. are weaker than covalent bonds

B. occur as a result of ionization

C. are the bonds formed between cysteines
D. stabilize secondary, tertiary, and quaternary

structures
7. With respect to lipid metabolism

A. fats are more reduced than carbohydrates
B. fatty acid synthesis occurs in the cytoplasm

C. ACP carries fatty acids across the inner mitochondrial

membrane

D. fat breakdown is stimulated by insulin
436
8. With respect to photosynthesis,

A. protons are pumped outside the chloroplast

B. oxygen is the terminal electron acceptor

C. ATP is produced by substrate level phosphorylation
D. oxygen is produced in the dark cycle

1. What does one have to remove in order to convert a weak acid
5. How does the pKa of a very weak acid compare to the pKa of a
weak acid ?
the pKa of a very weak acid is higher than the pKa of a weak acid
6. Explain precisely the piece of information that one would
always know if one had the Km for an enzyme.
the substrate concentration at Vmax/2
7. Name the strongest force stabilizing tertiary structure
disulfide bonds
into a salt?
a proton
2. Which amino acid in the catalytic triad of chymotrypsin gains a
proton transiently during the catalytic cycle?
histidine
3. Explain why myoglobin does not exhibit cooperativity
it only has a single subunit
4. Define cooperativity
when binding of one molecule to a protein favors binding
additional molecules of the same type by the same protein
8. Which strand in DNA replication contains the Okazaki

fragments?
lagging strand
9. What is the name of the sequence a few bases before the E.
coli (bacterial) translational start site?
Shine-Dalgarno site
10. What enzyme present in the developing embryo builds the
ends of the linear eukaryotic chromosomes?
telomerase
437
11. What trait commonly associated with a plasmid allows a

researcher to easily identify which cells get the desired DNA
16. When cells are going through fermentation, what molecule

are all of them trying to produce?
during transformation?
NAD+
antibiotic resistance
12. What is the name of the enzyme that is essential for removing
primers in E. coli DNA replication?
DNA Polymerase I
13. What protein in translation is responsible for translocation?
17. Define feedback inhibition

when the last molecule in a metabolic pathway inhibits the first
enzyme in that pathway
18. Besides glycogen, what other molecule is the substrate of
glycogen synthase?
EF-G
14. What is on the 5 end of eukaryotic mRNAs that is not on the
5 end of prokaryotic mRNAs?
a cap structure
15. Draw the Fischer form of D-glucose and its enantiomer (6
UPD-glucose
19. What enzyme of gluconeogenesis is inhibited by F2,6BP?
fructose-1,6-bisphosphatase (F-1,6BPase)
20. Besides acetyl-CoA, what is the only other 2 carbon molecule
in the glyoxylate cycle?
points)
glyoxylate
21. What enzyme catalyzes the big bang of glycolysis?
D-Glucose =
Enantiomer =
pyruvate kinase
22. Which molecule of the electron transport system is referred to
as the trac cop?
coenzyme Q (CoQ)
438
23. Name the molecule that directly donates two carbons to the
growing fatty acid chain.

malonyl-ACP
24. What enzyme of the cholesterol biosynthesis pathway is
1. One liter of a buer from the blood of Modest Mouse singer

Isaac Brock is found to have a pH of 7.2. The pKa of this buer
is 7.4. Does the buer have more salt or more acid?
feedback inhibited?
more acid
HMG-CoA reductase
25. Besides palmitoyl-CoA, what other molecule listed in class is
a precursor of sphingolipids?
Use the Henderson-Hasselbalch equation to justify your answer.

Your answer MUST be based in the Henderson-Hasselbalch
equation. Other answers will automatically be wrong. (7 points)
7.2 = 7.4 + log{[salt]/[acid]}
serine
26. What molecules are substrates for ribonucleotide reductase
Thus -0.2 = log{[salt]/[acid]}, so there much be more acid than salt
in making deoxyribonucleotides (answer must be precise)

27. When you develop gout, what molecule is precipitating in
2. Describe the sequence of events involved in the elongation of

translation in prokaryotes. Name the relevant proteins, RNA
molecules, and structures described in class and the functions
your cells?
they perform. (9 points)

uric acid
Three elongation factors of which two were named in class 28. Name an anti-cancer drug discussed in class that mimics
EF-Tu (carries proper amino acid to A site of ribosome and
folate
checks to be sure there is proper pairing between the anticodon

methotrexate
of the tRNA and the codon of the mRNA. Removes tRNA if

pairing not right. Also protects tRNA-amino acid bond from
water.)
439
EF-G - uses energy of hydrolysis of GTP to move ribosome one
Which direction will the reaction go when there are equal amounts
codon farther down the ribosome
of reactant and product? (Your answer MUST be based in the G
Sequence - tRNA-AA bound by EF-Tu enters A site of ribosome.
equation) (6 points)
If codon-anticodon pairing is proper, EF-Tu cleaves GTP and
The G value above is negative. Thus when there are equal
leaves. Otherwise EF-Tu removes tRNA-AA from A site. When
amounts of reactant and product,
proper tRNA in A site, 23S rRNA catalyses a peptide bond

between the amino acid attached to the tRNA in the P site and
G = negative number + RTln{1/1} = negative number + 0
the amino acid on the tRNA in the A site. Newly bonded structure
Consequently , G = negative number so reaction will move
is left linked to the tRNA in the A site. EF-G uses energy of
forward
hydrolysis of GTP to move ribosome one codon further in the 3

direction. This leaves the tRNA-peptide structure in the P site and
the A site is left open. The previous tRNA that was in the P site
moves to the E site and exits the ribosome.
3. A reaction has twice as much product as reactant at
equilibrium. What is the value of the G? ( I am not looking
for a number here) (2 points)
G = -RTln{2/1}
440
4. Compare and contrast electron transport and oxidative

phosphorylation to the light reactions of photosynthesis with
respect to electron sources, electron acceptors, movement of
protons, and any products of these reactions.
Photosynthesis
ETS/Oxidative phosphorylation
electron source - water
electron sources - NADH and FADH2
electron acceptor - NADP+ (makes NADPH)
electron acceptor = oxygen, making water.
splitting electrons and protons from water generates molecular

oxygen as a byproduct
NAD+ and FAD are byproducts
protons are pumped into the thylakoid space of the chloroplast by
protons are pumped out of the mitochondrial matrix to the
electron movement during the light reactions. ATP is made by
intermembrane space of the mitochondrion by electron
movement of the protons out of the thylakoid space through the
movement during electron transport. ATP is made in oxidative
ATP synthase
phosphorylation by movement of the protons back into the

mitochondrial matrix through the ATP synthase (Complex V).
Jump to Chapter
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
441
Chapter 12
Juicy Tidbits
Juicy Tidbits
Miscellany
Due to space restrictions, we werent able to include several
songs adjacent to the relevant text and we didnt want to leave
them out entirely, so we include them here starting on the next
page for your enjoyment.
One last plug :-)
We appreciate your support (financial or otherwise) to say thanks
for our eorts. They include buying a calendar (HERE), buying
The Splendidest Ever Metabolic Melodies songbook of lyrics
(HERE), registering for our ecampus classes at Oregon State
University (HERE, HERE, HERE, and HERE), and/or sending us a
message telling us how youve used the book. You can also join
and LIKE our Facebook page for Biochemistry Free and Easy
HERE.
Our email addresses are
kevin@davincipress.com
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You can view our biochemistry courses as follows below:

BB 350 - HERE / Download syllabus HERE
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We have a Web site full of Metabolic Melodies and Limericks
that you may enjoy. The URL is
http://www.davincipress.com/metabmelodies.html
Kevin released a book of Limericks in early 2013. More
information about that HERE. He also a few YouTube music
videos he is proud of. They include an anti-smoking video called
I Lost a Lung (from smoking Camels) and is to the tune of I
Left My Heart in San Francisco The link is HERE. The Oregon
rain gets noted in Let It Rain (to the tune of Let It Snow)
HERE, Around the Nucleus (to the tune of Across the
Universe) is HERE, and the OSU Alma Mater (to the tune of
Carry Me Back to OSU) HERE.
443
Ode to Bicarbonate
Translation
To the tune of Maria - from West Side Story
To the tune of "Song Sung Blue"
Translation
The most intricate thing I ever saw
From five prime to three prime, translation, translation
H-2-O
Ionizes slowly
You should know
Its behavior wholly
It presides - in cells insides - a solvent great

But mix it with an oil and shake it up
Youll see em separate
See em separate
C-O-2
Made in oxidation
Inside you
Decarboxylation
So divine when it combines up with the H-2-Os

Bonding together makes bicarbonates
To ease the pH woes
Bicarbonate can conjugate and take protons out

Restoring a balance inside of the blood
Of this there is no doubt
Thus its so
Thanks to bicarb buffer
In blood flow
You dont have to suffer
Bicarbonate will conjugate to take the protons out

Restoring a balance inside of the blood
Of this there is no doubt
Protons stow
In the bicarb buffer
Never grow
So you do not suffer
(fade)
The final step that we know about the central dog-ma

Amino, carboxyl, translation, translation. . . .
Translation, translation, translation . .
Translation!
I just learned the steps of translation
And all the things they say
About tRNA
Are true
Translation!
To form peptide bonds in translation
The ribosomal cleft
Must bind to an E-F
tee-you!
Translation!
A-U-G binds the f-met's cargo
16S lines up Shine and Dalgarno
Translation
I'll never stop needing translation
The most intricate thing I ever saw
Translationnnnnnnnnnnnnnnnnn


444
Chromatin
Good Protein Synthesis
To the tune of Sunshine on My Shoulders

Charges of the lysines bind to phosphates
Minus charges cling to plusses tight
Chromatin assembly is essential
Eukaryotic cells must get it right
Cells are tiny micro-scaled enclosures

With nuclei tucked deep in their insides
Thats the place amazingly enough that
Seven feet of DNA resides
H2a and H2b have lysines

Thats how they get charges dont forget
Paired with Hs three and four they make up
A chromatin cores octameric set
These get organized in higher orders

Changing with the cycles of the cell
Denser packing going through mitosis
At other times the structures simply swell
So because of all the hyperpacking

Nuclei can hold entire genomes
Thank the histones spooling DNA for
Physically downsizing chromosomes
To the tune of "Good King Wenceslaus"

Amino acids cannot join
By themselves together
They require ribosomes
To create the tether
All the protein chains get made

Cording to instruction
Carried by m-R-N-A
In peptide bond construction
Small subunit starts it all

With initiation
Pairing up two RNAs
At the docking station
Shine Dalgarnos complement

In the 16 esses
Lines the A-U-G up so
Synthesis commences
Elongation happens in
Ribosomic insides
Where rRNA creates
Bonds for polypeptides
These depart the ribosome

Passing right straight through it
In the tiny channels there
Of the large subunit
Finally when the sequence of

One of the stop codons
Parks itself in the A site
Synthesis cant go on
P-site RNA lets go

Of what it was holding
So the polypeptide can
Get on with its folding


445
Transcription
To the tune of Frosty the Snowman
Phos-pho-di-esters
Are the bonds of RNA
That support a ribopolymer
Made of G,C,U and A
Histones
The RNA polymerase

Binds to a Pribnow box
And copies from the template strand
All the along the way it walks
To the tune of "Meet the Flintstones"

Histones, tiny histones
IN-i-ti-a-tion
Of transcription thus proceeds
From the closed to open complexing
In the DNA it reads
Wrap up eukaryotic DNA
The sigma factor gets released

Its work is over fast
Polymerase can then advance
After this step has been passed
Using lysine side chains

They arrange a chromatin array
In elongation
The polymerizing spree
Moves along the way in fits and starts
Synthesizing five to three
With them - DNAs of seven feet

Fit in - side the nucleus so sweet
The RNA is floppy and

It dangles from one end
Oh thats a most important thing
For you to comprehend
When you use the histones
You have to deal with condensation
Then termination
Fin-ish-ES the RNAs
Thanks to protein rho or hairpin forms
That release polymerase
And its ablation

Inside your chromosomes
So this describes transcriptions steps

In three part harmonies
Heres hoping with this melody
You (can) learn it all with ease


446
The Bloody Things
Structural Lullaby
To the tune of Coke It's The Real Thing
To the tune of "Brahms' Lullaby"
Im gonna put some oxygens
In your sleep
You can keep
Learning more about sugars
Fischer schemes
Haworth rings
D & L and everything
Hydroxides
Cant collide
Favring chair over boat form
Spatial guides
Coincide
With the way structures form
beside my porphyrin rings

To nudge the irons up a notch
and yank on histidines
The globins shapes will change a bit,
oh what a sight to see
The way they bind to oxygen
co-op-er-AH-tive-ly
And as I exit from the lungs

to swim in the bloodstream
Metabolizing cells they all
express their needs to me
To them I give up oxygen
and change from R to T
While my amines, they hang onto
the protons readily
But that's not all the tricks I know,

there's more thats up my sleeve
Like gaps between sub-U-nits that
hold 2,3-BPG
When near metabolizing cells,
I bind things that diffuse
The protons and bicarbonates
from lowly cee oh twos
CHORUS
Thats the way it is
When your cells are at play
Go say hip hip hooray
For the bloody things

Recording by David Simmons

447
The New Serine Protease Song
My Old Enzymes
To the tune of Rudolph the Red-Nosed Reindeer
To the tune of "Auld Lang Syne"
You know threonine, cysteines and trusty aspartates

Cobalts, magnesium and glutamates
But do you recall
The best characterized enzymes of all?
All serine proteases
Work almost identically
Using amino acid
Triads catalytically
First they bind peptide substrates

Holding onto them so tight
Changing their structure when they
Get them in the S1 site
Then there are electron shifts

At the active site
Serine gives up its proton
As the RE-ac-tion goes on
Next the alkoxide ion

Being so electron rich
Grabs peptides carbonyl group
Breaks its bond without a hitch
So one piece is bound to it

The other gets set free
Water has to act next to
Let the final fragment loose
Then its back where it started

Waiting for a peptide chain
That it can bind itself to
Go and start all oer again
Wheneer my proteins go kaput

If they are past their prime.
The cells will act to soon replace
All of my old enzymes
They know which ones to break apart

Ubiquitins the sign
A marker for pro-TE-a-somes
To find the old enzymes
These soon get bound and then cut up

In pieces less than nine
More chopping yields the single ones
Building blocks from old enzymes
So in a way the cell knows well

Of father time its true
Amino acids when reused
Turn the old enzymes to new
T
448
In Closing . . . . . .
The End
To the tune of The End
The End
Hoc est finis

This is the end
Of Free and Easy Biiiiiii-o-chem

Copyright
Information
Biochemistry Free and Easy 2013 Kevin Ahern & Indira Rajagopal
All rights reserved
& Disclaimer
This is version 2.0 of this electronic book. It is also version 2.0 of the PDF form of this
book.
Disclaimer
Every eort was made to ensure that information contained in this publication was as
accurate as possible at the time of publication (July 24, 2012). However, Kevin Ahern
and Indira Rajagopal make no claims that the information contained anywhere in this
publication is, in fact correct, so users assume responsibility for all ways in which they
use the information herein. This publication is therefore provided as is and all
responsibility for use of information herein resides solely on the user. Further, Kevin
Ahern and Indira Rajagopal make no claims about medical validity and oer no medical
advice regarding anything stated in this books nor to hyperlinks to any other content
provided here. Kevin Ahern and Indira Rajagopal oer no advice of any sort. Anyone
seeking medical or other advice needs to consult medical or other relevant professionals
for such advice.
Wikipedia Licenses
Figures from Wikipedia were used under the Creative Commons or GNU license and are listed
according to their URLs as follows.

Page 13 Onion cells - http://en.wikipedia.org/wiki/File:Wilson1900Fig2.jpg
Page 14 Extremophile - http://en.wikipedia.org/wiki/File:Grand_prismatic_spring.jpg
Page 14 Water/Iceberg - http://en.wikipedia.org/wiki/
File:Iceberg_with_hole_near_sanderson_hope_2007-07-28_2.jpg
Page 36 Light reactions photosynthesis - http://en.wikipedia.org/wiki/File:Z-scheme.png
Page 43 Amino acid - http://en.wikipedia.org/wiki/File:AminoAcidball.svg
cdl
Page 45 Protein structure - http://en.wikipedia.org/wiki/File:Main_protein_structure_levels_en.svg

Page 46 Beta sheet - http://en.wikipedia.org/wiki/File:Beta-meander1.png
Page 46 Tropocollagen - http://en.wikipedia.org/wiki/File:Collagentriplehelix.png
Page 48 Ramachandran plot http://en.wikipedia.org/wiki/File:Protein_backbone_PhiPsiOmega_drawing.jpg
Page 48 Myoglobin - http://en.wikipedia.org/wiki/File:Myoglobin-1mba.png
Page 50 Water on leaf - http://en.wikipedia.org/wiki/File:Water_drop_on_a_leaf.jpg
Page 57 Prions - http://en.wikipedia.org/wiki/File:Prion_propagation.svg
Page 60 A,B, Z DNA - http://en.wikipedia.org/wiki/File:A-DNA,_B-DNA_and_Z-DNA.png
Page 61 Supercoiling - http://en.wikipedia.org/wiki/File:Circular_DNA_Supercoiling.png
Page 63 tRNA - http://en.wikipedia.org/wiki/File:TRNA-Phe_yeast_1ehz.png
Page 68 Oligosaccharide - http://en.wikipedia.org/wiki/File:Branched_oligosaccharide_struct.svg
Page 70 Glycogen - http://en.wikipedia.org/wiki/File:Glykogen.svg
Page 70 Cellulose - http://en.wikipedia.org/wiki/File:Cellulose_Sessel.svg
Page 70 Glycosaminoglycan - http://en.wikipedia.org/wiki/File:Hyaluronan.png
Page 79 - Vitamin D - http://en.wikipedia.org/wiki/File:Cholecalciferol-3d.png
Page 99 - Ribozyme - http://en.wikipedia.org/wiki/File:Ribozyme.jpg
Page 108: Proofreading - http://commons.wikimedia.org/wiki/File:DNA_polymerase.svg
Page 119: T7 RNA polymerase - http://commons.wikimedia.org/wiki/File:T7_RNA_polymerase_at_work.png
Page 144 - Glycolysis - http://commons.wikimedia.org/wiki/File:Glycolysis.svg
Page 182 - Calvin Cycle - http://en.wikipedia.org/wiki/File:Calvin-cycle4.svg
Page 189 - Trp metabolism - http://en.wikipedia.org/wiki/File:Tryptophan_metabolism.png
Page 199 - Nerve junction - http://commons.wikimedia.org/wiki/File:Neuron_synapse.png
Page 205 G-protein pathway - http://commons.wikimedia.org/wiki/File:G-protein.svg
Page 207 PKC activation - http://commons.wikimedia.org/wiki/File:Activation_protein_kinase_C.svg
Page 218 - DNA fragment separation - http://commons.wikimedia.org/wiki/File:Gel_electrophoresis_2.jpg
cdli
Page 219 - SDS-PAGE separation of proteins - http://en.wikipedia.org/wiki/File:SDS-PAGE.jpg
The End
To the tune of The End
Hoc est finis

This is the end
Of Free and Easy Biiiiiii-o-chem

cdlii

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Dedication & Credits

Biochemistry Free and Easy is dedicated to

Dr. E.C. Nelson

Dr. George Pearson

Biochemistry Free and Easy 2012, 2013

All images credited to Wikipedia have license information on the Copyright

Casey Luce &

Dr. Amber Leis

Dr. Anna Hsu

Dr. Bory Kea

Dr. AnneMarie Corgan

Dr. Chrissy Lamun

Jessica Page with

Jenny Tran, Andrea

Dr. Kyle Shaver

Dr. Laura Miller

Dr. Marie Strand

Matt Lewis and

Sarah Ferrer & Emily

Paul Jones and

Dr. Sara Haidar

Dr. Tara Humphrey

Theresa & Maria Nguyen

Thi Nguyen, Justin Biel &

Dr. Tom Sharpton

David Simmons, Liz

life with their delightful

We are indebted to several

us could ever dream of

doing. David is currently a

medical student at Oregon Health

State University student who created, or oversaw the creation of,

Biochemistry and Biophysics (link

HERE) at Oregon State University

following URL - www.mjbakerartist.com. She was assisted by

(link HERE) where we teach

biochemistry, molecular biology and Honors biology to hundreds

provided by a faculty grant to Kevin from the L.L. Stewart fund

supporting faculty eorts that improve teaching at OSU.

biochemistry called Metabolic Melodies,

We have a Web site full of Metabolic Melodies

many of which were used here.

and Limericks that you may enjoy. In the spirit

We decided when we created this book

that information should be as freely

support (financial or otherwise) to say

thanks for our eorts. Ways to do this

Kevin has also released a book of limericks titled

include buying a calendar (HERE) or the

A Limerick A Day For A Year More

The Splendidest Ever Metabolic

information about that HERE.

classes at Oregon State University (HERE, HERE, HERE, and

He also a few YouTube music videos he is proud of. They include

HERE), and/or sending us a message telling us how youve used

an anti-smoking video called I Lost a Lung (from smoking