Rough Schedule:: Questions? Please Ask Along The Way!

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Omid Vadpey
Bio98 Quiz 1 Review
Rough Schedule:
Useful techniques for Critical Thinking questions (10 mins)
Energetics of reactions(30 mins)
Enzymes and Reaction Rates (25 mins)
pH of Amino acids, Functional Groups (30 mins)
3D Structure and Function of Proteins (25mins)
*Evaluations will be passed out towards the end of the review.
Questions? Please ask along the way!
*Pictures and concepts in the following presentation have been cited from the Lehninger textbook and
-Biochemistry-Lectures Copyright Pavan Kadandale and Lehninger et al- All rights reserved.
Francesco
Nguyen
Preparing to Think Critically
1.
2.
3.
4.
5.
6.
Actively read and understand concepts

Ask Questions
Research, discuss (yourself + others)
Applications
Practice Questions
Have fun!
Approaching Critical Thinking Questions

1.
2.
3.
4.
Differentiate between givens and unknowns

a.
Whats significant?
Draw or write to relate concepts to the problem
Justify your conclusion with those concepts
Smile
Example Problem: Energetics of Reactions

Under standard conditions, ATP hydrolysis (G) is
-30.5 kJ/mol. Use the resulting plot below to
explain why metabolism is regulated to keep the
ratio of ATP/ADP high in the cell.
AT
P
0.2
AD
P
0.2
2.2
4.2
5.0
25
Using Our Techniques
Under standard conditions, ATP hydrolysis (G)

is -30.5 kJ/mol. Use the resulting plot below to
explain why metabolism is regulated to keep the
ratio of ATP/ADP high in the cell.
ATP IS IMPORTANT: coupled with countless
reactions in the cell!
Energetics of reactions
Before we answer that question, lets do a little review.
Delta G: Free Energy Change
Delta G (G):
energy change given in
varying conditions (cell =
dynamic)
function of temperature,
concentrations
Represented by:
G=G+RTln[(prod/react)]
Q= [Product]i/[Reactant]i
Direction the reaction will go
given all conditions
Standard Delta G (G):

tells us nature of products and
reactants
direction reactants want to go
at standard conditions
Represented by:
G= -RTln(Keq)
Ratio of products and reactants AT
EQUILIBRIUM
Demonstration:
ATP-->ADP + Pi
VS. (MORE) ATP ADP+Pi
Concept 1: one way to increase magnitude (-)G is

increase intracellular reactant concentrations.
APPLICATION = Glucose (blood)
Back to Our Problem:

Under standard conditions, ATP
hydrolysis (G) is -30.5 kJ/mol. Use the
resulting plot below to explain why
metabolism is regulated to keep the ratio
of ATP/ADP high in the cell.
ATP ADP+ Pi
ATP
(m
M)
0.2
AD
P
0.2
2.2
4.2
5.0
25
Answer?
The cell wants to keep ATP at a
high concentration so that
because ATP hydrolysis is lower
when there are more products
than reactants. Thus, energy
available to the cell is higher
when ATP/ADP ratio is greater.
G=G+RTln[(prod/react)]
Given: standard delta G <0
RTln(<<1) <0 -->More reactants
RTln (>>1) >0 less reactants,
less potential energy (delta G is
smaller)
Recap
Understand context of a cell versus

standard conditions
Be able to think about multiple
scenarios
QUESTIONS?
One more Delta G
If the reaction A+B C has a G= -25 kJ/mol. Which

molecules will be at the highest concentrations at
equilibrium?
A. Molecule A
B. Molecule B
C. A and B
D. Molecule C
E. Cannot be determined. We dont know other conditions
Questions?
Last G Problem
Consider the hypothetical transformation of
X Y, for which G= 10.0 kJ/mol
a. what is the ratio of [Y]/[X] at equilibrium?
b. If you add a molecule Z, that reacts with Y, what will happen to the G of X
Y?
c.
The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction

in a timely fashion (seconds rather than days). If you add an inhibitor to block
the function of YA, what will happen to your products in that time?
Answer 1:

. Ans: X!
a. If you add a molecule Z, that reacts with Y, what will happen to the G of X
Y?
b. The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction
Answer 2

. Ans: X!
Y?
. Ans: G will become more negative, goes forward
a. The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction
Answer 3

. Ans: X!
Y?
. Ans: G will become more negative, goes forward
a. The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction
. Ans: it will take a while before you see any product, but once at equilibrium,
ratio will be the same!
Energetics: Coupling Reactions
Is it feasible to only increase concentrations in cells?

No!
Concept 2: Thats why we couple delta G!
coupling transport (membranes)
gradient formation
glucose transport in intestine
Biochem reactions, and more
Energetics: Coupling Reactions
Is it feasible to only increase concentrations in cells?

No!
Concept 2: Thats why we couple delta G!
coupling transport (membranes)
gradient formation
glucose transport in intestine
Biochem reactions, and more
Your favorite reaction (so far)
1) Glucose + Pi glucose-6-phosphate +H20

G= 13.8 kJ/mol
2) ATP +H20 ADP+Pi
G= -30.5 kJ/mol
3) Sum: Glucose +ATP glucose-6-phosphate +ADP
G sum = -16.2 kJ/mol (thermodynamically favored forward)
Enzymes
Do enzymes shift the equilibrium?

o
No, enzymes do NOT shift the equilibrium
So what can they do?

o
All they can do is SPEED UP the reaction
How do they do that?

o
o
by LOWERING the Ea (activation energy) orient molecules in a way that makes them
easier to react
stabilizes T.S.
They are SPECIFIC!

o
What accounts for the SPECIFICITY of an enzyme?
its functional group diff. interactions
Enzyme Active Sites
What determines the enzymes interaction with its

substrate?
o
o
Am. Acid residues of the active site (functional groups)

Enzyme shape (defined 3D structure)
Active site residues initiate rxns and stabilize TS

CHIRAL and sensitive to chirality
NOTE!
Enzymes CANNOT change whether a reaction is favorable or not.
All an enzyme will do is:

o
make the reaction more kinetically favorable
o
by lowering the Ea of the rxn
ex. if a reaction doesnt proceed because its not favorable ( G
=+)
o adding an ezyme will not make the reaction go
Enzyme Problem
Suppose an enzyme undergoes a mutation that no longer

allows it to form a conformation change. How will this affect
a reaction?
Will the rxn occur?
o
no, a conformational change is needed for the substrate to bind to the enzyme (perfect,
specific fit)
What would happen to the rate of the reaction?

o
undergoes normal rate (wont speed up, bc no enzyme-substrate complex forms)
no Ea lowered
Enzyme Problem 2!
Lets say an active site is made up of amino acid residues

that are all in a line. However, these residues now become
spaced out throughout the enzyme (could be due to a
mutation).
How will this affect the enzymes ability to bind the
substrate?
o it wont be able to bind to its normal substrate
What does it have to do in order to bind?
o
o
can bind with a chaperone protein

Chaperone proteins stabilize enzymes so it wont mutate/denature
Enzyme Problem 3!
For the conversion of glucose to G6P, hexokinase is used.

If more hexokinase was injected into the body, how would
this change the delta G of the reaction?
The delta G is not affected because it is independent
from [enzyme]
Enzyme Problem 4 (last one)
To speed up the rate of a glucose rxn, enzyme WAT is

used. To speed up rate of galactose reaction, enzyme SUP
is used. How would you speed up the rate of a lactose (glc
+ galac) rxn?
adding WAT/SUP wont work
you need to find a SPECIFIC enzyme for lactose
Looking at lecture, why cant our body digest L-Glc but it
can digest D-Glc?
Hexokinase SPECIFIC to D-GLucose! active sites
are CHIRAL and sensitive to chirality
Amino Acids, and Functional Groups

1.
2.
3.
Amino acid groups (sticky note)

pH/Titration curves
Calculating overall charge
Why are pH curves important?

Maybe a few reasons:
1.
Structure = function (extremely critical) any change in pH or temperature leads to _____
2.
Knowing pH isoelectric points tells us:
a.
Types of interactions
b.
Location in protein/cell
c.
biochemical modeling
Modeling of Proteins
location?
sequence?
bonds?
o
interactions
charges
Common Questions
1.
Should we know the name and structures of all the amino acids?
At least know: groups they are categorized in (hydrophobic, polar, charged)
Amino Acid Groups: Sample Question
Name three amino acids that could

be located in the hydrophobic
region of a transmembrane
protein?
What about the amino acids in the
inside of a ionic channel in
neurons?
Answer:
Sample Question 2
What is the isoelectric point of cysteine?
K2
Protein/pH Question
You and a group of friends in your research lab decide to

purify a secretion protein that is important in innate immune
defense. This class of protein normally has positively
charged amino acids such as histidine and arginine. Thus,
you decide to purify it using a negatively charged resin in
the hopes to eventually figure out the entire sequence. At
this point, the buffers were added with a pH of 10 and a salt
concentration of 300 mM NaCl and the lysed solution was
run through the resin. VERY little protein was collected.
Why?
Whats a better way to purify the protein from the bacterial cell?
Questions?
After successfully purifying your defensive protein, you and

your lab partners hypothesize where the protein is secreted
and used in the body.
Ideas?
Mucous membranes of the intestine
Saliva
Skin
Respiratory mucous membranes
What is the overall charge of a protein that has the following sequence at pH=7?
Glu-Gly-Ala-Lys
Which charge resin could you purify this with?
Positive, Negative, you cant?
3D Structure and Function of

Proteins
Anfinsens Experiment
What did he want?

o
Wanted to unfold a protein
So what did he do?

o
he heated the protein
denatured H bonds/weak bonds
What happened?
o
Proteins lost activity
How a protein folds

is determined by
PRIMARY
STRUCTURE!!
He left it to cool over time. What happened?

o
Protein refolded (deduced that bc protein activity came back over time)
Jeoffrey
(Activity)
Cersei
(Activity)
Nothing
400
380
Heat
22
26
Heat +
Recovery
379
29
Heat +
Recovery
+ Jamie
379
368
Heat +
Jamie
26
28
Heat +
Jamie +
Recover
378
30
Given the following

hypothetical enzymes
and observations, what
could be said about the
enzymes, Jeoffrey,
Cersei, and Jamie?
Jeoffery is capable of refolding
himself. Cersei cant. Cersei
requires the chaperone protein,
Jamie in order to refold. Jamie
cannot fold by itself.
Primary Structure
Protein folding information contained in the primary am.

acid seq.
Made up of peptide bonds (planar)
o
can rotate due to resonance
Secondary Structures (3D)
A - helix
o
formation of H bonds stabilizes struc. by orienting hydrophobic regions outwards
o
this stabilizes the struc bc if outward, they would have constant replusion with water
gives them a mobile struc and its struc is specific to protein function
o
use of internal H-bonds (between C & N terminus backbone) causes a-helix to form helix
struc
o
R- groups parallel
Secondary Structures
B- pleated sheet
o
allows the structure to function fully in a zig zag and alternating sequence
H-bonds can interact with each other laterally in an anti confomation

o
parallel/anti-parallel
Tertiary Structure
Hydrophobic interactions (H bonds)/weak bonds

determines the folding of tertiary structures
o
driving force for folding
combines A & B structures
Problem!
If a cell were going to create a protein that exists within the

plasma membrane, how would you expect the protein to
fold?
It would fold so that its hydrophobic amino acids are
facing outwards so that it is able to interact with the
nonpolar fatty acid tails that make up the interior of the
plasma membrane
And thats it!

QUESTIONS!?!? Ask now :)
Extra Resources:
Office hours on Tuesday, day before your exam.
Check: https://sites.google.com/a/uci.edu/biotutor/

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Bio98 Quiz 1 Review

Preparing to Think Critically

Actively read and understand concepts

Approaching Critical Thinking Questions

Differentiate between givens and unknowns

Example Problem: Energetics of Reactions

Using Our Techniques

Under standard conditions, ATP hydrolysis (G)

Delta G: Free Energy Change

Standard Delta G (G):

VS. (MORE) ATP ADP+Pi

Concept 1: one way to increase magnitude (-)G is

Back to Our Problem:

Understand context of a cell versus

One more Delta G

If the reaction A+B C has a G= -25 kJ/mol. Which

The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction

Consider the hypothetical transformation of

Consider the hypothetical transformation of

Consider the hypothetical transformation of

Energetics: Coupling Reactions

Is it feasible to only increase concentrations in cells?

Energetics: Coupling Reactions

Is it feasible to only increase concentrations in cells?

Your favorite reaction (so far)

1) Glucose + Pi glucose-6-phosphate +H20

Do enzymes shift the equilibrium?

No, enzymes do NOT shift the equilibrium

So what can they do?

All they can do is SPEED UP the reaction

How do they do that?

They are SPECIFIC!

What accounts for the SPECIFICITY of an enzyme?

its functional group diff. interactions

Enzyme Active Sites

What determines the enzymes interaction with its

Am. Acid residues of the active site (functional groups)

Active site residues initiate rxns and stabilize TS

Enzymes CANNOT change whether a reaction is favorable or not.

All an enzyme will do is:

ex. if a reaction doesnt proceed because its not favorable ( G

Suppose an enzyme undergoes a mutation that no longer

What would happen to the rate of the reaction?

undergoes normal rate (wont speed up, bc no enzyme-substrate complex forms)

Lets say an active site is made up of amino acid residues

can bind with a chaperone protein

For the conversion of glucose to G6P, hexokinase is used.

Enzyme Problem 4 (last one)

To speed up the rate of a glucose rxn, enzyme WAT is

Amino Acids, and Functional Groups

Amino acid groups (sticky note)

Why are pH curves important?

At least know: groups they are categorized in (hydrophobic, polar, charged)

Amino Acid Groups: Sample Question

Name three amino acids that could

You and a group of friends in your research lab decide to

After successfully purifying your defensive protein, you and

3D Structure and Function of