Biochemistry

Proteins and amino acids

Myoglobin - O2 transporter in muscle.

Has heme
Lots of protein globulin
Polar residues exterior, non-polar residues interior
Amino acid composition - primary structure of protein
Amino acids are connected by peptide (amide) bonds
Naturally occurring 20 amino acids
Example: protein collagen has some modified a.a. hydroxylysine and
hydrohyproline; Vit.C is responsible for the hydroxylation
Proteins can be also modified by phosphorylation
Ph
at Ph7 H = 10 and OH = 10
at Ph3 - [H+] = 103 and [OH- ] = 1011
Lysine and arginine are + charged at neutral ph
Aspirin has a COO group at ph~4 (like in duodenum) and a COO+ at more acidic ph
~2 (stomach); absorbed easier when is uncharged.
Isoelectric point such ph at which there is no charge present
Henderson-Hasselbach equation know the relationship
Chaperons aid in protein folding
Saturation curves for hemoglobin:




Hemoglobin contains 4 subunits, once oxygen binds to one subunit others have higher
affinity allosteric modification
Low ph, high CO2 and high DPG lower hemoglobin affinity for oxygen
In sickle cell anemia there is a substitution of Val to Glu on the beta chain






Enzymes

Many enzymes are proteases
Digestive enzymes are released in the zymogen (inactive) state and then cleaved and
become active; example trypsinogen is cleaved by enterokinase to become trypsin
Protease inhibitors inactivate the proteases
Serine proteases have serine at the active site
Thrombin is a serine protease, it is attached to the endothelial membrane
Mechanism of action of most enzymes - increase the speed of the reaction by lowering
free energy of the transitional state









Inhibition:
Competitive inhibitors bind at the same site, increase Km
Noncompetitive inhibitors
Uncompetitive inhibitors binds to the enzyme substrate complex, change both Km
and Vmax
Irreversible inhibitors





Allosteric interaction binding at a different site of the enzyme causes the active site
to bind better; example hemoglobin
Know what is free energy, entropy, enthalpy (change in heat content), equilibrium
constant
Often reactions with unfavorable free energy (positive) are coupled to the ones with
favorable free energy (negative); example glucose to glucose 6 phosphate is coupled with ATP
hydrolysis.


Metabolism (please refer to the hand out)


In di- and monosaccharides glucose can be linked by different types of bonds (a- 1,4
glucose in starch, which is digestible; and -1,4 glucose in cellulose which is not digestible)
Ribulose important in ribosemonophosphate shunt
Xylulose in photosynthesis
Complete oxidation of 1 gram of carbohydrates gives 4 kilocalories
Amylase breaks down starch
Intestinal enzymes lactase, maltase, isomaltase, sucrase act at the surface of the
absorbing cell
Fructose is taken in the cells by a carrier mediated passive transport
Glucose and galactose are taken in by a sodium linked active transport; there is always
a sodium gradient, created by sodium potassium ATPase
Liver regulates the glucose level in blood it takes the glucose during the meal and
releases it later to maintain glucose level
RBC can not survive without glucose because they do not have mitochondria, brain
can use ketone-bodies
Glucose transporters in the muscle and fat are regulated by insulin, in intestine and
kidney there are active transporters
Glycolysis is a continuos process, occurs in the cytoplasm
In the absence of oxygen glucose goes through the glycolysis pathway in the
cytoplasm which ends in pyruvate or lactate
Glycolysis yield total of 2 molecules of ATP: 4 molecules per glucose are produced
and 2 molecules are used
Liver regulates the glucose level in blood it takes the glucose during the meal and
releases it later to maintain glucose level
RBC can not survive without glucose because they do not have mitochondria, brain
can use ketone-bodies
Glucose transporters in the muscle and fat are regulated by insulin, in intestine and
kidney there are active transporters
NAD is a coenzyme in glycolysis, NAD and NADH are involved in the oxidation
reduction processes
Enolase is a specific target for the fluoride intoxication
Regulated enzyme of glycolysis PFK phosphofructokinase which responds to
changes ATP and ADP but the key regulator in the body is the fructose 2,6 bisphosphate
Pyruvate kinase is another regulatory enzyme
In RBC lactate is the end product, formation of lactate regenerates NAD needed for
the previous steps of glycolysis
Glycolysis is a universal process
Pyruvate goes on to the TCA cycle and high amount of ATP are generated through
oxidative phosphorylation in the mitochondria
Glucose can be synthesized in the liver from small precursors - lactate, pyruvate,
amino acids, glycerol, but not from fatty acids breakdown product acetyl Co-A
First step in gluconeogenisis (reversal of the glycolysis) pyruvate to oxaloacetate
occurs in the mitochondria; needs a coenzyme derived from biotin
Unique steps of gluconeogenesis go around irreversible steps of glycolysis, example
PFK, hexokinase
Gluconeogenesis occurs in the liver, kidney and to a small extent in the intestine
Insulin and glucagon are the regulator
Glucagon always leads to the phosphorylation of proteins
Glucagon will increase blood sugar level
Glucose 6 phosphate is used in pentose phosphate shunt
1
st
enzyme is glucose-6 phosphate dehydrogenase which uses NADP
NADPH protect against oxidative damage, used in synthesis of fat and steroids
Pentose phosphate pathway provides pentoses
Extra steps are required for the metabolism of fructose and galactose to convert them
in to the intermediates of glycolysis
Sugar nucleotides are used in the synthesis of polysaccharides
UDP glucose is used in the formation of glycogen
Glycogen has a-1,4 links with a-1,6 links at branching points
Glycogen synthase lengthens the chain, then there is a branching enzyme forms a-1,6
links
Breakdown of glycogen: glycogen phosphorylase and a debranching enzyme
Regulated steps are synthase and phosphorylase
Addition of phosphate has an opposing effect on these 2 enzymes, it deactivates the
synthase and activates the phosphorylase
Key regulators insulin and glucagon

Citric acid cycle and oxidative phosphorylation

TCA occurs in mitochondria
Pyruvate is converted to acetyl Co-A :pyruvate + CoA + NAD = acetyl Co-A + CO2 +
NADH
Acetyl Co-A enters the TCA cycle, where NADH, FADH and GTP are produced
NADH and FADH are used in electron transport chain
Oxydative phosphorylation occurs in the inner mitochondrial membrane
Electron transport chain generates a proton gradient across the membrane which is
used to generate ATP

Fatty acid metabolism


Animals can not convert fat to glucose
Triglycerol (3 fatty acid residues) main reservoir of fat
Triglycerol is broken down by lipase in to 3 FA and glycerol
Nomenclature of fatty acids: 2 different systems COOH group carbon is designated
as delta carbon or the last carbon on the other end as omega carbon
Fat is broken down in two units yelling acetyl Co-A which goes in to TCA cycle or
production of ketone bodies
During starvation ketone bodies are produced; can be used by the brain as a source of
energy
Fatty acid synthesis also occur in 2 carbon units, except in the first step malonyl Co-A
is added
Regulated step of the synthesis is acetyl Co-A to malonyl Co-A which uses biotin as a
coenzyme

Cholesterol synthesis


Acetyl CoA + acetoacetylCoA = HMG CoA
HMG-CoA + NADPH = melavonate + NADP this is the first commited step.
Regulated by the enzyme HMG-CoA reductase
Statin molecules inhibit this enzyme, statins are used in many cholesterol lowering
drugs
Cholesterol is used to make bile acids, biological membranes, steroid hormones
and Vit.D
Cholesterol in bile acids is secreted in the small intestine and later is reabsorbed,
transported back to the liver and recycled
LDL is a major cholesterol carrier, contains apoprotein ApoB which is specific for the
liver cells
After a cholesterol rich meal, liver cell contains a lot of cholesterol esters, which
inhibits HMG reductase and down regulates LDL receptors on the liver cell membrane
LDL enters liver cell by receptor mediated endocytosis
Hypercholesterolemia can be caused by defect in the downregulation of HMG
reductase by cholesterol in the liver cell
Or due to downregulation of the LDL receptors in the liver which leads to high LDL
levels in the blood
High LDL in the blood causes LDL uptake by endothelial cells in the blood vessels as
well as other tissues

Amino acid metabolism

Essential amino acids come from the diet
Know what they are
Precursors of a.a. synthesis:
Oxaloacetate aspartate
Pyruvate alanine
Alpha keto glutarate glutamate
3 phosphoglycerate
Transaminase enzyme acts in the transfer of the amino group to alpha ketoglutarate
Tetrahydrofolate is used in one carbon units transfer
Urea cycle
BIOCHEMISTRY REVIEW (PART 2)


Signaling

Hormones
Tree chemically distinct classes of hormones: amino acids (or amine hormones),
peptides and steroids
peptides TRH,ACTH,ADH, insulin and glucagon
Amines epinephrine and tyroxine
Steroid cortisol, aldosterone, estradiol, testosterone,progesterone
Steroid hormones are derived from cholesterol, so that inhibition of HMG-reductase
by cholesterol lowering drugs can lead to inhibition of steroid production
Another group are locally acting hormones like prostaglandins, leukotriens and
thromboxanes
Peptide hormones are fast acting, steroids are slow acting with long lasting effect
Insulin is secreted as preproinsulin with a signal sequence at one end and c-peptide in
the middle, it is proteolytically processed to form mature insulin
Prostaglandin synthesis is inhibited by aspirin
There are multiple isoforms of cyclooxygenase, aspirin inhibits all, including one that
acts in the stomach to produce a type of prostaglandin responsible for mucus production, that is
why aspirin is a stomach irritant
New drug was developed that does not act on cyclooxygenase in the stomach lining

Hormone receptors

G-protein linked receptors
Tyrosine kinase receptors
Serine/Treonine receptors
Steroid and thyroid hormone receptors in the nucleus
Gs-protein adenylate cyclase c-AMP protein kinase A phosphorylates proteins
Different effects in different cells:
Ex: epinephrine binds to R in liver, everything to protein kinase A is the same, protein
knase A in liver activates glycogen phosphorylase kinase, which activates glycogen phosphorylase
and inhibits glycogen synthase; in smooth muscle protein kinase A acts on myosin light chain
kinase inactivates myosin, causing relaxation.
G-protein linked R have the same basic structure, all span the membrane 7 times
Clinically relevant example: cholera toxin covallently modificates Gs protein by ADP
ribosalating it which blocks its GTPase activity, so that G-protein stays constantly active
Phosphodiesterase is responsible for the breakdown of c-AMP; caffeine inhibits
phosphodiesterase
Another type of G-protein is Gq protein
Gq protein acts on phospholipase C which breaks down PIP2
(phosphoinositolpyrophosphate) in to IP3 (inositol tri phosphate) and DAG (diacylglycerol)
IP3 and DAG are second messengers
IP3 stimulates Ca release, Ca acts on Ca/calmoduline dependent protein kinase
Calmoduline is a Ca binding domain in Ca dependent kinases
Breakdown of IP3 is inhibited by lithium
Tyrosine kinase R - growth factors, insulin
Tyrosine kinase R phosphorylates itself after hormone binding
Autophosphorylated tyrosine kinase R becomes a binding site for SH2 domain of
proteins, a cascade follows which leads to activation of RAS protein
RAS is a GTP binding protein
In many cancers there is a mutation in RAS protein which prevents RAS from
hydrolyzing back to GDP (inactive form) so that RAS is always on
RAS activates RAF kinase kinase cascade stimulates cell growth
Serine/Threonine kinases - receptors for bone morphogene proteins



Nucleic acid structure and synthesis

Nucleotide nitrogenous base, pentose, phosphate
Nucleoside nitrogenous base and pentose
Nucleotides building blocks for nucleic acids
Genetic info is carried in the sequence of nitrogenous bases; two types of bases
purines (A and G) and pyrimidines (C,T and U)
A double bonds to T
G triple bonds to C
In RNA T is substituted by U
DNA contains deoxyribose hydrogen atom instead of hydrogen group on carbon 2
Phosphate groups form the linkages between nucleotides
3`-5` phosphodiester linkage is the most common; binding always occurs in 5` to 3`
direction
DNA replication occurs by base paring
DNA polymerase:
Synthesis in 5` to 3` direction
Uses antiparallel template
Associated with proofreading enzymes
Requires a primer short strand of RNA complementary to the template strand
Helicase forces two parental strands of DNA to separate at the replication fork
Topoisomerase unwinds 3-dimentional structure of DNA
DNA gyrase winds up DNA to package more efficiently
Synthesis of one of the daughter strands of DNA occurs continuously (leading strand)
Other strand (lagging strand) is made in short fragments (Okazaki fragments) that are
then joined together
Okazaki fragments are also synthesized in the 5` to 3` direction

Transcription

RNA polymerase
Specific sequences in DNA act as start and stop sequences
Promoter sequence binds RNA polymerase allows for start of transcription
Steroid hormone receptor complexes bind to enchancer sequences on DNA which
activate promoter sequences
3 steps : initiation, elongation, termination
inhibitors of prokaryotic transcription:
rifampin inhibits initiation
actinomycin D blocks movement of RNA polymerase
RNA is initially synthesized as a precursor molecule containing introns and exons
Introns are spliced out
Processing of the RNA includes 5` capping, polyadenylation and RNA splicing

Techniques in molecular biology

Restriction endonucleases are enzymes that recognize specific double-stranded
sequences of DNA and cleave the DNA at the restriction site
Know about hybridization techniques
Sothern blot
Nothern blot
PCR polymerase chain reaction is used to multiply DNA fragments provided you
have primer sequences

Protein synthesis

mRNA messenger RNA acts as a working copy of a gene coding for a protein
rRNA component of ribosomes
tRNA bring amino acids to the site of protein synthesis, they have anticodone region
which is complementary to the codone of the mRNA
tRNA`s react with amino acids at their 3` end
Specific amino acyl synthesases attach amino acids to t RNA
Start codone on the mRNA is AUG coding for methionine
Translation factors are GTP binding proteins that bring amino acid tRNA complexes
to ribosome
Look at the table in the book for list of antibiotics that interfere with translation
Dipthteria toxin - ADP ribosalates a GTP binding protein that is involved in
translation , inhibiting translation




Signal sequences

Dolichol carrier for surgar molecules that are added to proteins in Goldgi