Biochemistry

Proteins and amino acids


Myoglobin - O2 transporter in muscle.

Has heme
Lot’s 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+] = 10¯3 and [OH- ] = 10־11
Lysine and arginine are + charged at neutral ph
Aspirin has a COOˉ 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 ß-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