1

Protein
PROTEIN STRUCTURE
• Protein is macromolecules (built up from repeating units of monomers/amino acids to form polymers)

• Formation of peptide bonds: peptide bonds joined by carboxyl group of 1 A.A with amide group of the
next A.A in the sequence.

4 Level of Protein Organisation

Primary structure, 1°
• Linear sequence of A.A residues joined through the peptide bonds to form polypeptide chain

• Function: all a.a have different side chain (polar/hydrophilic , non-polar/hydrophobic, acidic.basic)

Secondary structure, 2°
• Recurring: formed by H-bonding & also rigid structure

- α-helices, β-sheets (parallel & anti-parallel) - if parallel is has same direction

- Function: rigid for the creating binding sites & for stable protein structure
• Non-recurring: no H-bond formation which usually found on protein surface

- Bends, loops, turns

- Function: flexible to allow segment of polypeptide chain to move as a compound binds or to move as
protein fold around another molecule. -

Tertiary structure, 3°
• Folding of 2° structure to 3D conformation (flexible and dynamic)

• Stabilised by H-bond, ionic bonds, van Der waals, interaction of hydrophobic effect, disulphide bond.

• Function: 3D structure specific and flexible binding sites for ligands and maintain residues for protein’s
cellular location (polar—> cytosolic residues while non polar—> transmembrane residue)

Quaternary structure, 4°
• Multi-subunit complex which the contact region between subunit is non-polar side chain, H-bond, ionic
bond and salt bridges

• Function: increased stability of protein, increased size and number of possible interaction between a.a
which cause difficult to unfold and refold. It also increase affinity binding sites for large molecule and
exhibit cooperatively between subunits in binding ligands

Structure-Function Relationship
Haemoglobin, Hb - 4° tetramer ( 2α & 2β pp chain)
• Alteration in Hb structure: change 1 a.a where hydrophobic valine takes place of hydrophilic glutamate at
β chain will alter the shape of Hb & creates sickle cell anaemia

- Alteration of RBCs shape which cause occluded capillaries, lack of blood flow through capillaries will
lead to hypoxia and tissue damage

Immunoglobulin, Ig - 4° tetramer ( 2 identical light & 2 identical heavy by disulphide bond)

• Variable domain (specific for antigen that is bound and can change shape) & constant domain (cannot
change shape)

- Alteration of RBCs shape which cause occluded capillaries, lack of blood flow through capillaries will
lead

Fibrous Globular

4°, simple, long and narrow elongated 3D structure Compact, highly coiled (ball-like structure), non-polar
(rope-like structure) toward the centre and polar toward the edge

Structural purpose Functional purpose

Generally insoluble in water Generally soluble in water

Eg: collagen, keratinin and fibrin Eg: insulin, Hb, myoglobin and enzyme

°. α, β, γ , δ
 2
PROTEIN DIGESTION AND ABSORPTION

Protein Digestion (protein—> amino acid)

In stomach
• Gastric parietal cells: secretes HCl to alters conformational of pepsinogen to pepsin, also partially
denature proteins and kill microorganism

• Gastric chief cells: secrete pepsinogen (zymogen) which able to cleave self (autocatalysis) with the help
of HCl to form pepsin (active protease pH~2)

- Pepsin cleaves peptide bonds within the protein chain to form smaller peptide and free a.a

By enzyme from pancrease

• As gastric content empty to intestine, they encounter secretion from exocrine pancrease.

• Pancreases will secrete HCO3- to neutralise acidity of stomach content and raise pH for pancreatic
protease enzyme to act

Pancreatic zymogen Active form Types of enzyme A.A specificity

Trypsinogen Trypsin Endopeptidase Cleaves peptide bond of carboxyl group. eg: lysine
, arginine

Chymotrypsinogen Chymotrypsin Endopeptidase Cleaves peptide bond of hydrophobic a.a

Proelastase Elastase Endopeptidase Cleaves elastin and peptide bond of small side
chained a.a

Procarboxy- Carboxypeptidase A
Exopeptidase Cleaves a.a from carboxy end of peptide chain at 1
peptidase Carboxypeptidase B time (A-Hydrophobic a.a while B- Lysine, arginine

By enzyme from intestinal cells

• Brush border of intestinal cells: secrete aminopeptidase- cleaves a.a at amino-end of peptide

• In epithelial cells: secretes intracellular peptidase- act on smaller peptides that absorbed by the cells

Amino Acid Absorption

Secondary active: Na+ dependent Co-transport
•At brush border membrane of epithelial cells, A.A transported to intestinal
cells together with Na+ driven by low intracellular Na+ concentration where
Na+ is pumped out in exchange with K+ by Na+-K+ ATPase.

Facilitated diffusion (bidirectional)

•At serosal membrane of epithelial cells into the interstitial fluid

•A.A can diffuse back into intestinal cell during starvation

Amino Acid Transportation

•A.A that enter the blood are transported into various tissue (same like
absorption) and have specific A.A transport system

•Eg: β° for basic amino acid (cystinuria) β°+ for zwitterionic A.A (hartnup
disease)

Protein Turnover

• Our body’s amino acids pool come from dietary amino acids and degradation of protein within the cells

• All cell & enzyme has 1/2 life. Eg: after t1/2 of protein, it will degrade to A.A for recycle

• How proteins within the cell degraded?

- Lysosomal protein turnover: autophagy process where protein is engulfed into lysosome and
cathepsin inside the lysosome degrades the proteins into A.A for recycle

- Ubiquitin-Proteasome pathway: ubiquitin targets intracellular proteins and proteasome will degrade
the targeted protein into A.A for recycle

Disease related to defect in protein digestion

• Hartnup disease: defect in transporting of neutral and essential a.a in intestine and cause malabsorption
while in renal tubule will reduce reabsorption, increased A.A in urine which lead to hyperaminoaciduria

- Clinical features: pellagra (light sensitive rashes, ataxia-lack muscle coordination, nystagmus )

• Cystinuria (autosomal recessive): disorder of a.a transporter cystine will cause inadequate cystine
reabsorption, increase cystine in urine which lead to cystinuria

- Clinical features: cystine stone in kidney, ureter and bladder

 3
AMINO ACID TRANSAMINATION, DEAMINATION & DECARBOXYLATION

Transamination (located at cytosol & mitochondria all tissue) - except lysine & threonine
• Nitrogen is transferred as amino group from original a.a to α-ketoglutarate forming glutamate whereas
the original a.a forming α-ketoacid
• By enzyme aminotransferase & transaminase and required cofactor which is pyridoxal phosphate (PLP)
as carrier of amino group to be transferred

Alanine (liver) Aspartate (liver, muscle, RBC)

Alanine aminotransferase (ALT) to measure diagnostic Aspartate aminotransferase (AST) to measure liver
liver function test damage/ cardiac marker
when liver cell is damaged, enzyme leaked

Enzyme Ilike into blood, if elevated can be normal if we Enzyme leak into blood, if elevated can be muscle injury,
do strenuous exercise or hepatitis and cirrhosis muscle injury and acute liver damage

Glutamate enter oxidative deamination while pyruvate and oxaloacetate will enter kreb cycle

Oxidative Deamination (located at mitochondria of liver and kidney) - glutamate

• Removal of a.a nitrogen as ammonium ion by glutamate dehydrogenase

• Required coenzyme NAD+/NADP+ and the product is α-ketoacid and ammonium ion/ NH4+

• Relation of oxidative deamination

- Fed state, increased insulin, ATP, GTP = allosteric inhibitor

- Fasting state, increased glucagon, ADP, GDP = activator of GDH

Oxidative Decarboxylation (located at mitochondria of most tissue) -methionine, threonine, valine,

isoleucine
• Removal of carboxyl group from α-ketoacid by enzyme α-ketoacid dehydrogenase

• Product is propionyl CoA & CO2 then propionyl will mtebolized into succinyl CoA and enter kreb cycle

Maple Syrup Urine Disease (valine, leucine and isoleucine)

• Disorder in oxidative decarboxylation due to defect in α-ketoacid DH enzyme

- α-ketoacid cannot decarboxylated will cause accumulation of α-ketoacid. This will lead to
neurological deterioration

• Clinical features:

- Burnt syrup smell urine & ketonuria, poor feeding, development delay and behavioural problems

AMINO ACID DERIVED COMPOUNDS (SLP)

Porphyrins (<— glycine)

• Found in many important compound including Hb, myoglobin and cytochrome

• Glycine + succinyl CoA —> α-aminolevulinate x 2 —> phorphobilinogen x 4—> porphyrin

Creatine (<—glycine + arginine + methionine) & Glutathione (<— glycine+glutamate+cysteine)

• Phosphocreatine derived from creatine which required 1 ATP

- Hydrolysis of phosphocreatine to creatine produces energy in muscle

• Glycine, glutamate and cysteine formed reduced glutathione (required 2 ATP) then it will undergoes
redox activity which link by disulphide bond to form oxidised glutathione

- Helps maintain sulfhydryl group of proteins in the reduced state & iron of heme in Fe2+ state

- Serve as reducing agents and redox function used to remove toxic peroxide formed

 4
Biological Amines are product of amino acids decarboxylation
• All of these pathway is a.a decarboxylation and required PLP *

• Tyrosine—> * Dopamine —> norepinephrine —> epinephrine

- Decreased dopamine will lead to Parkinson’s Disease

- Increased dopamine will lead to schizophrenia

• Glutamate —> * γ-aminobutyrate (GABA)

- Decreased GABA will lead to epileptic seizures

- GABA analog can be used for treatment epilepsy and hypertension

• Tyrptophan —> serotonin

• Histadine —> histamine (powerful vasodilator in animal)

- For allergic response & stimulate acid secretion in stomach

• (Methionine + ATP —> decarboxylated adoMet) + (ornithine —> putrescine) —> spermidine

• Spermidine + decarboxylated adoMet —> spermine

- Spermidine and spermine involve in DNA packaging

• Ornithine decarboxylaase, PLP requiring enzyme is powerful inhibitor used as pharmaceutical agents

Purine and Pyrimidine

• Purine derived form glycine

• Pyramidine and purine also derived from aspartate and glutamine

Amino acid metabolism is important for fuel metabolism, disposal of toxic substances, synthesis of
protein and polypeptides.

FATE OF NITROGEN & CARBON SKELETON OF AMINO ACID

Fate of Nitrogens
• Nitrogen enters the urea cycle as NH4 from oxidative deamination (in mitochondria) and aspartate from
glutamate by transamination to oxaloacetate (in cytosol)

Urea cycle (mainly in mitochondria of liver & intestine)

• Conversion of toxic
NH4 (toxic to brain
& CNS) to less toxic
urea, then
transported in the
blood for excretion

1.Formation of
carbonyl phosphate

2.Formation of
citrulin

3.Formation of
argininosuccinate

4.Cleavage of
argininosuccinate

•Fumarate is
converted to malate
which is used for
synthesis of glucose
or for regeneration
of oxaloacetate

5.Cleavage of
arginine

• Urea is derived from aspartate, NH4 and CO2

 5
Fate of urea
• Urea diffuse from liver into blood & then into kidney where it is excreted as urine

• Some urea diffuse into small intestine to be cleaved by bacterial urease to form CO2 & NH3-

- This ammonia/ NH3- will lost in faces or reabsorbed into the blood

Regulation of urea cycle ↑ ammonianon-purenation

• Substrate availability: if high rate of ammonia production, high rate of urea formation

• Allosteric activation of CPSI by N-acetylglutamate/NAG (NAG synthesis stimulated by arginine)

I
- In fed state,Iincreased arginine in liver,1increased synthesis of NAG, increased synthesis of
CPSI,x increased rate of urea cycle1
x Arg + r synthesis
• Induction/repression of the synthesis urea cycle enzyme
N lasthesis taste aycle
of

- IHigh protein diet./ prolonged

. fasting,
. increased protein metabolism, increased rate of urea
cycle

Metabolism of Ammonia
protein
↑

metabolism,
t
↑
ayrere
rate.
Sources of ammonia
• Transamination and oxidative domination of amino acids

• From glutamine by renal/intestinal glutaminase

• Reaction on urea by intestinal bacterial urease release ammonia (absorbed into portal vein & converted
to urea in liver)

• Dietary amines & monoamines are reacted by amine oxidase

• Purine and pyrimidine degradation

Transportation of ammonia (ammonia always low in blood)

• Alanine and glutamine are carrier of ammonia in blood to liver

- [Alanine in muscle 1
-> alanine
- Glutamine in muscle and peripheral tissue
carry NH3
eauseoperakaiver
-
->
carry
Disorder of Urea Cycle
wits
• Accumulation of ammonia in the circulation are toxic to brain and CNS

• Under normal conditions, ammonia is rapidly fixed into α-ketoglutarate or glutamate or by CPSI

• When urea cycle enzyme is defective, the cycle will interrupted which lead to an accumulation of urea-
cycle intermediates & &lead to glutamine accumulation, decreased α-ketoglutarate. -
1 This leading to high
S
level of ammonia in blood

• Ammonia toxicity

- Brain
->
swelling (+osmotic imbalance) which caused by high level of ammonia & glutamine in
astrocytes
+
cerebral oedema -> brain
- High level of ammonia will inhibit glutaminase cause toswelling
increase production of glutamine (alter
permeability of mitochondria which may lead to death)

- High level of ammonia, decreased glutamate (neurotransmitter) which lead to impaired

---
-
neurotransmitter thus causing lethargy and reduced nervous system activity.

Brain swall
-
-> inhibit glutaminase
-> glutamine prod, of
&fifammonia
-
idea
toglutamate i
permeability atter
mitochondri
#

↓N Sactivity.
&

c)
i lethargy-impaired auto
transmitter
-
 6
Fate of Carbon Skeleton

Amino acid synthesis and degradation

• Total of amino acid is 20 which has unique, diverse, different properties and supply nitrogen

- 9 essential a.a: body cannot synthesis and must obtain from by diet

- 11 non-essential a.a: body can synthesis

• Degradation of amino acid to

- CO2

- Compound that produce glucose in the liver (α-ketoglutarate, pyruvate, succinyl CoA, fumarate &
oxaloacetate) considered as glucogenic a.a

- Ketone bodies or their precursor (acetoacetate & oxaloacetate) considered as ketogenic a.a

• Ketogenic A.A: isoleucine, threonine, tyrosine, tryptophan, phenylalanine

• Both ketogenic and glucogenic A.A: lysine and leucine

• Glucogenic A.A: other than that

Glucose -Alanine cycle

•In muscle

- Glucose undergoes glycolysis to form

pyruvate

- Pyrauvate is transaminated by glutamate

to form alanine

- Alanine is transported into blood then to

liver

• In liver

- Alanine is transaminated to form pyruvate

and nitrogen

-Nitrogen used for urea production

-Pyruvate used to produce glucose through

gluconeogenesis

-Glucose then is transported to muscle

Nitrogen balance (protein contain 16% of N)

• Milk secretion is major source of N loss in lactating women

• Postive N balance (intake>output) , increased in total body protein - normal

- During growth [hase in infants & young children, pregnancy and wound healing

- During athletes and periods of anabolic training

• Negative N balance (output>intake , loss of body protein - not normal

- When people under stress, patient with various injuries, trauma, burns, surgery and infection, dietary
deficiency of 1/more essential a.a

• Blood urea nitrogen can be used in estimating N-balance

• Obligatory N loss from body when no protein consumed or a.a continuously degraded

- If protein intake < obligatory N loss, amount of N excreted > amount of N consumed which can be
considered as negative N balance state

• Use k-jeldahl reaction to measure total nitrogenous compound in food, faeces and urine

 7
SEMINAR
Homocystinuria (autosomal recessive illness)

• Increase plasma & urine level of homocysteine, methionine & decreased level of cystine

• Deficiency of cysthationine-β-synthase and decreased in folic acid, cobalamin and pyridoxine

• Methionine <— homocystine —> cystathionine

Phenylketonuria (autosomal recessive illness)

• Decreased metabolism of phenylalanine & accumulation of phenylalanine in blood, tissue and urine

• Deficiency of phenylalanine hydroxylase (chromosome 12)

• Defect on dihydropteridine reductase

• Symptoms: mousey odor, mental retardation by age of 1 and hypo-pigment

Parkinson’s Disease (autosomal recessive illness)

• Dopamine function as a neurotransmitter in the brain & as a local chemical messenger outside the CNS

• Free radical attacks will disrupt and destroy the dopamine synthesise cells causing deficiency of
dopamine

Albinism (autosomal recessive illness)

• Defect in TYR gene at 11 chromosome

• Lack of melanocytes, tyrosine hydroxylase to produce dopa

• Symptom: absent pigment from skin and eyes

Nucleic Acid
PURINE, PYRAMIDINE, NUCLEOSIDES, NUCLEOTIDE
• Nucleic acid (formed by condensation & polymerisation reaction) - repeating unit of nucleotide. Eg: DNA
& RNA that encode genetic information

-
1 is N-glycosidic bond (between
F C1 & base)

Type to enter text 2 is phosphodiester bond (between

* C5 & phosphate group or C3
& phosphate group of new nucleotide

• Phosphate group: mono-P, di-P, tri-P

Is
• Sugar molecule: if ribose has -OH on c2 while deoxyribose has -H on C2

• Nitrogenous base:

- Purine (dicyclic ring): adenine, guanine (have =O)

- Pyramidine (monocyclic ring): cytosine, thymine (have =O & methyl group), uracil (have =O)

• Structure
->nucleoside no phosphate
- LNucleoside: nitrogenous base + sugar by N-glycosidic bond.- Eg: adenosine/ deoxyadenosine or
cytidine/deoxycytidine
-> nucleotide ada phosphate
- LNucleotide: nitrogenous base + sugar + phosphate by N-glycosidic bond and phosphodiester bond.-
Eg: adenosine-5-triphosphate (ATP), cAMP (cyclic nucleotide - phosphate group is bounded to 2
sugar’s hydroxyl groups forming cyclical / ring structure)

- INucleic acid: polynucleotide by phosphodiester bond 3

of previne
a pyrimidine
STRUCTURE OF DNA & RNA pairing
Structure of DNA (composed of 2 strands & base pair) ↳ G-.- 2
4 A... T
• Concept of base pairing
w
- L2 strand of DNA joined by H-bonds- 4
between the bases
IH bond
- Purine- - -pyramidine. If A- - -T used 2 H-bond while G- - -C used 3 H-bond
3 Abond
- 2 strands of DNA are complementary
- which
. allows one strand of DNA to serves as a template3for the
- . -
-
synthesis of the other strand & also synthesis of a1complementary strand of RNA

• DNA strands are antiparallel which 2 complementary strands of DNA run in opposite direction.

- 5’-3’ (sense strand) whileO

0-. 3’-5’ (anti-sense strand)

5.3 vs 35 is sentiasa opposite

• LDouble helix3( twisting of 2 DNA strand)

- Base pairs that joined the 2 strands are stack, which stabilised by van Der Waals interaction and
hydrophobic effect, H-bond of base pairs

- 3rd OH- group on the phosphate is free & dissociate a H+ at physiological pH, each DNA helix has
negative charges coating on its surface that facilitate the binding of specific protein

• Major and minor groove

- Major groove: more open,Irich in chemical information7 and protein can recognised the .
DNA sequence

- Minor groove: more constricted, T

- .

-
poor
I in chemical information and less useful

• Linear & circular DNA (have both: !bacteriophage in varion particle)

⑪
- Linear DNA molecules in eukaryotic cell

- Circular DNA molecules in monkey DNA virus, bacteria E.coli and plasmid

cell cox
E
oi <
cell
venkary acteria
linear
f
y

p
lasmid
virus
DNA -
I DNA
circular monkey
 9
Structure of RNA (single strand, ribose sugar, lack of continuous helical structure, 2° or 3° structure)

- Base pair are determined by DNA via transcription

• Structure of mRNA

- Transcribed from protein-coding

genes

- Precursor: pre-mRNA/ hnRNA

- mRNA consist of

-i
- Leading sequence: at L5’-end begin withIguanosine cap

- Coding region + begin with start codon that signals the beginning of translation, followed by
-w -

trinucleotide codon for a.a and end with stop codon

- Trailer sequence: terminates at its 3’-end with poly(A)tails

• Structure of rRNA
- Cytoplasmic ribosomes contain 2 ribosomal subunit (40S and 60S) while mitochondria ribosomes
(55S) smaller than cytoplasmic ribosomes

• Structure of tRNA (cloverleaf shape)

- Contain 2 structure: stem (H-bond) and loop (non-bond). Some loop contain modified bases which
are dihydrouridine, pseudouridine, ribothymidine.

• Structure of miRNA
- Small RNA molecule that regulate (reduce) protein expression at post-transcriptional level by induce
degradation of a target mRNA or block translation of the target mRNA.

Physical Properties of RNA & DNA

• Denaturation (separation process of double helix DNA)

- pH : in acidic, DNA & RNA will degrade while in alkaline, DNA will denature and RNA will degrade

- 2 strand of DNA will separate but does not break phosphodiester bond

- Phosphodiester bond of RNA are cleaved because OH- on C2 losing its proton, allowing -ve carted
oxygen to attack & break phosphodiester linkage

- Heat (melting temperature Tm = 50% of DNA separated) : when Tm high, increased concentration of
salt, Na+, K+ and DNA concentration
-

...

• Hybridisation (renaturation @ reannealing)

- After heated, slowly cooled then single stranded DNA anneals/meet with complementary of DNA @
RNA sequence

- Annealing temp: between 45°-65°

• UV absorbance
- DNA & RNA maximally absorbs UV light at wavelength 260nm due to base (sugar-phosphat does not
contribute to UV absorption)

- When temp is high, DNA denatured, wavelength more than 260nm ‘hyperchromicity’, single strand of
DNA are more exposed and has high capacity of bases to absorb UV compare to double strand DNA.

 10
Synthesis of purine and pyramidine can be through
(
• De novo pathway (occur in the liver, brain, neutrophils and other cells of immune system) : synthesis from
low molecular weight precursors

• Salvage pathway (occur within liver or lymphocytes) : synthesis from nucleosides or bases that become
available through the diet or from degradation of nucleic acids (reutilisation)

PURINE NUCLEOTIDE METABOLISM

• Building blocks for DNA & RNA, only purine-pyramidine pairs fit inside the double helix

• Energy currency, carriers for activating intermediate, structural components and singling molecules

Purine biosynthesis (9 atoms in purine)

• Precursor (5) for N is glutamine, aspartate and glycine while C is glycine, N10-formyl tetrahydrofolate and
CO2

• Food that contain high purine: anchovies, clams, beef, lobster, salmon

De Novo pathways -> in livel

• Synthesis of inosine monophosphate (IMP) - (10 steps, required 6 ATP, branch point, precursor of AMP &
GMP, contain base hypoxanthine)

- Ribose-5-phosphate + ATP —> 5-phosphoribosyl-1-amine pyrophosphate (PRPP) by PRPP

synthetase

- PRPP + glutamine —> 5-phosphoribosyl-1-amine by glutamine phosphoribosyl transferase /

amidophosporibosyl transferase (this enzyme is inhibited by IMP, AMP & GMP)

• Synthesis of adenosine monophosphate (AMP) - (2 step)

- IMP + GTP + aspartate —> adenylosuccinate by adenylosuccinate synthetase

- Release fumarate from adenylosuccinate —> AMP by adenylosuccinate lyase

• Synthesis of guanosine monophosphate (GMP) - (2 step)

- Hypoxanthine base in IMP (+NAD+) is oxidised —> xanthine monophosphate (XMP) by IMP DH

- XMP + glutamine + ATP —> GMP

Phosphorylation of AMP & GMP to Ldi- & tri-phosphate levels.3 Triphosphate for RNA synthesis

Reduction of ribonucleoside DP to deoxyribonucleoside DP (dADP, dGDP) occur only for DNA synthesis
- -
->
and required NADPH, thioredoxin, thioredoxin reductase.
- reduction
resulting in formation of
• Regulation of purine synthesis
ATP, DPTPS used for DNA
D
- CPRPP synthetase3 is inhibited by ADP and GDP
synthesis.
- Glutamine phosphoribosyl-amidotransferase is inhibited by GMP & AMP

- Adenosuccinate synthase is inhibited by AMP

- IMP dehydrogenase is inhibited by GMP

- If GTP high, AMP will be synthesised while if ATP high, GMP will be synthesised.

Salvage pathways -> lymphocyte

• Nucleotide (AMP) can be reconverted from
adenosine by adenosine kinase (require ATP)

• Free base (hypoxanthine & guanine) can be reform

from nucleoside (inosine & guanosine respectively)
by purine nucleoside phosphorylase reaction

• Hypoxanthine-guanine phosphoribosyltransferase
(HGPRT) reaction will recover hypoxanthine /
guanine to use for resynthesis of nucleotide

• APRT reaction will recover adenine to AMP

Purine Degradation (harm the body if too high)

•Excreted as urate

•Some metabolic disorder- gout,

hyperuricaemia, immunodeficiency
disease

 Synthesis of IMP.
5-phosphates
1.

ribose

PRER
ATP
Hatalyzeby
CPRPPC
ssphosphoribosyl 1-pyrophosphate
-

e
H2O
OClutamine phosphoro 1St
CIntamipe-

5-
phosphoribosylamine
synthetase
O
ATP GAR
Glycine added
Glycinamide ribonucleotide (GAR)

GAR
transformulaso

(FGAR)

FGAM
↓
synthetase.

CFGAMS

S
1Mp (firstpurinenucleotidare
 (synthesis of adenosine (AMP
behove
pathway monophosphate

addedsuccinate fumerate
farmarate is

aspartate is
3 adenylossucinate
* released from
IMP +
IMP to
adeny
↓x by adenylsuccinate) AMP
atp
2s f
rotate
AT Adenplosuccinate
GDP + pi

CAMPS

Be
pathway (synthesis of guanosine monophosphate
novo (MPC.

watanoctionophosphate
IMP

wodipinnedre oa
e

->
glutamine donate
amide to
nitrogen
xanthine
monophosphate (XMP) AMP) Amp
ATP)
Crequires ·
 11
PYRAMIDINE METABOLISM
• Uracil (in RNA), thymine (in DNA), cytosine (both) orotic acid / orotate (intermediate)

Pyramidine synthesis (6 atoms in purine)

• Precursor (2) : carbamoyl phosphate and aspartate

• Pyramidine are not synthesis as nucleotide but as pyramidine ring (orotate) then it attached to ribose-5-
phosphate to form nucleotide

De Novo pathways
• HCO3- + glutamine (NH3-) + 2ATP —> carbomyl phosphate by CPS II

• Carbomyl phosphate + aspartate —> carbomyl-aspartate by aspartate transcarbomylase (comitted step)

• Carbomyl-Asp —> dihydroorotate —> orotate + PRPP —> orotidine monophosphate (OMP) —> uridine
monophosphate (UMP) by OMP decarboxylase

- UMP + ATP <—> UDP + ADP by UMP kinase

- UTP + ATP + glutamine —> CTP (to RNA) —> dCTP (to DNA)

• Regulation of pyramidine synthesis

- Carbomyl phosphate inhibited by high level of UTP and activate by PRPP

- Aspartate transcarbomylase (ATCase) inhibited by CTP and activated by ATP

- Regulated by cell cycle.

Salvage pathways

Production of Deoxyribonucleotide (reduction of RNA —> DNA)

• Reduction occur in di-phosphate level which catalysed by ribonucleotide reductase which required
thioredoxin, thioredoxin reductase and NADPH

• Ribonucleotide reductase will activated/on if ATP while it will inhibited/off by dATP

Pyramidine Degradation (not harm the body if too high)

• In some organism not human, free pyrimidines are salvage & recycle to form nucleotide
phosphoribosyltransferase reaction

• In human, pyramidine are recycle from nucleoside but free pyramidine bases are not salvages

• Cytosine or uracil —> β-alanine, NH4+, CO2

• Thymine —> β-aminosobutyric acid, NH4+, CO2

De nove
pyrimidine synthesis
CPSI

HCOsGlutamine
capu parate
->
Yutamate

lie
os

ATP H2O + 2 ADP +#

noplaces
+ 2 +

<
Part 1.

part ↳
i
carbaquaringtarouse
dihydroorotate
dihydroorotate treatmonies
dehydrogenase
of
ump

&
Klo2
onaarboxylase.
- part
3.

Orotidine
sn -

5
Orotate

*
orotate

phosporibosyl
transferase!
monophosphate
COMP)
 12

Molecular Biology Genetics I

DNA REPLICATION
1. DNA replication occur in S phase of cell cycle and begins at the point of Origin (oriC) & occur at two
replication fork which leading to local separation of strand

2. This separation is facilitated by:

• Helicase: unwinding the double helix

• Single-strand binding proteins (SSBs): keep the two strand separated and prevent premature re-
annealing of dsDNA

3. As two strand of double helix separate, it will lead positive supercoils which are removed by:

• DNA topoisomerase: enzyme that break the phosphodiester bond & rejoin them)

4. Local separation of strand allowing binding of DNA polymerase which synthesis new DNA strand only
in the 5’-3’ direction (anti-parallel). DNA polymerase require RNA primer

5. Each strand of double helix serves as template for constructing 2 complementary daughter strand (a
process called semiconservative reaction)

6. Consisting of

• 1 leading strand: synthesised continuously in 5’-3’ direction toward the replication fork which require
I

only one RNA primer

• 1 lagging strand: synthesised discontinuously in 5’-3’ direction away from replication fork which require
many 1 RNA primer

7.
Prokaryotic - Ecoli Eukaryotic
• One origin of replication • Several origin of replication
• DNA chain elongation catalysed by
• DNA pol α + primase = RNA primer

- DNA pol III: add nucleotide and • DNA pol ε is to complete DNA synthesis on leading strand while
also proofreading of newly DNA pol δ elongates the Okazaki fragment of the lagging strand

synthesis DNA by removing • Both ε & δ use 3’-5’ exonuclease activity to proofread

terminal mismatched • DNA pol β involve in DNA repair while DNA pol γ replicated
nucleotides with it 3’-5’ mitochondria DNA

exonuclease activity
• RNA primer removed by FEN1 and RNAse I

• DNA pol II involve in DNA repair

• Telomeres are complex located at the end chromosomes,
• RNA primer are removed by
problem happen in lagging strand bcs telomeres shorten so
- DNA pol I: using it 5’3’ telomerase (contain both RNA & protein) act as reverse
exonuclease activity and also transcription / as RNA-dependent DNA pol by using RNA
fills the gaps with DNA template to synthesis DNA in usual 5’-3’ direction extending
3’overhang

8. The final phosphodiester linkage is catalyse by DNA ligase which join the 2 nucleotides together
(3 OH- gp at the end of 1 fragment is ligated to the phosphate gp at the 5-end of the next fragment)

9. Termination: terminus utilisation substance (TUS) to perlication termination sites on DNA, it will stop the
movement of DNA pol

DNA repair
• Action of mutagen

- Chemical produced in the cell. Inhaled/ absorbed from environment: Benzo(a)pyrene

- Radiation: X-rays (hydroxyl radical that alter the structure), UV rays (it will form pyrimidine dimer)

- Infectious agents: HPV nad H.pylori

Repair mechanism
• Distortion in DNA helix is recognised & removed. Gap in damaged strand replaced by the action of DNA
pol or by undamaged strand as template. Ligase will stealth nick in strand that undergone repair

• Nucleotide excision repair: involve local distortions of DNA helix and specific repair endonuclease
cleave the abnormal chain

- Xeroderma pigmentosum (sun-induced skin abnormalities): inability to remove UV induced thymine

dimers in the DNA and lead to mismatch during DNA replication through thymine dimer

• Base excision repair: DNA glycosylases recognise small distortion in DNA and cleaves the glycosidic
bond that joined damage base

 13
• Mismatch repair: mismatched bases are recognise by enemies of mismatch repair system

- Hereditary non-non-polyposis colon cancer (HNPCC) due to mutations in intestinal enzyme

• Transcription-coupled repair: gene that are actively transcribed to produce RNA are repaired by
excision repair protein

- Cockayne syndrome (premature aging) : cell cannot transcribe damaged gene, DNA cannot be repair
due to defect in transcriptional-coupled repair

DNA replication inhibitor: cytosine, arabinosine

DNA TRANSCRIPTION (DNA/GENOMIC —> RNA)

Transcription of Prokaryotes
1. 1 RNA pol synthesis all different types of RNA (mRNA, tRNA, rRNA)

2. σ factor (sigma) bind to the core enzyme enable RNA pol to recognise promoter region on the DNA

3. Process transcription can be divided into 3 phase

• Initiation:

- Transcription begins with binding of RNA pol holoenzyme (σ factor+ core enzyme) directly to a
promoter region on DNA to form preinitiation complex (PIC)

• Elongation:

- σ factor is then release cause conformational changes of the core enzyme & core enzyme slides on
DNA template toward 3’ end

- Free nucleoside triphosphate (NTPs) are added to 3’ OH of nascent RNA strand

- RNA pol + DNA segment of ~40 nucleotide + nascent RNA forming transcriptional bubble

- 3’ segment of nascent RNA hybridises with DNA template and it 5’ end extend out transcriptional
bubble a synthesis is processing

• Termination : RNA stop moving on DNA template by

- RNA fold back on itself forming hair pin loop in transcription preceding a number of 4 residues

- Ρ factor (rho) an ATP dependent helicase it will disrupts and separates nascent RNA-DNA complex

Transcription of Eukaryotes
1. 3 RNA pol synthesis different types RNA

• RNA pol I (rRNA), RNA pol II (mRNA, miRNA), RNA pol III (tRNA, rRNA)

2. Process transcription can e divided into 3 phase

• Initiation:

- Chromatin remodelling binding of at least 6 transcriptional factor only then RNA can bind to promoter
region of DNA up/down stream coding region facilitated by specific TF bound to enhancer sequence

- Promoter for gene transcribed by RNA pol II that contain characteristic of consensus sequences
(TAAT-TATA or Pribnow box)

• Elongation:

- Requires local unwinding of DNA helix followed by synthesis of 5’-3’ RNA transcript coded for by the
DNA template that lead in 3’-5’ direction

• Termination :

- Requires termination signal sequence result in release of RNA pol & newly synthesised transcripts
from DNA

Prokaryotic mRNA = primary transcript

Primary transcript for both prokaryotic & eukaryotic are post-transcriptional modified by cleavage of
original transcript by ribonuclease

Post-Transcriptional Modification of RNA

(splicing of pre-mRNA to removed non-dosing introns & join exon)

• rRNA
- Pre-rRNA (long precursor) cleaved by ribonuclease then trimmed by exonucelases and modified at
some base or sugar

- Prokaryotic rRNA: 23S, 16S & 5S rRNA

- Eukaryotic rRNA: 28S, 18S, 5.8S rRNA

• tRNA (1binding site for specific sequence of 3 nucleotide in mRNA while 1binding sites for encoded a.a)
- Pre-tRNA, removal of intron by nucleases & both end trimmed by ribonuclease

- Modified at some base and 3’-CCA sequence is added

 14
• mRNA (eukaryote only) - primary transcript = hnRNA
- Capping at the 5’end: 7-methylguanosine cap (to decrease rate of degradation; serves as recognition
site) attached to 5’ end through 5’-5’ linkage

- Tailing at the end 3’end: a long poly A tail is attached to 3’ end

- mRNA splicing: slicing of pre-mRNA to removed non-coding inions and join exon to produce mature
mRNA

DNA transcriptional inhibitor: antimyosin D (block RNA pol), rifamipicin (inhibit RNA pol), distamycin
(alter transcriptional)

DNA TRANSLATION (RNA —> PROTEIN/ PHENOTYPE )

(the genetic message encode in DNA is 1st transcribed into mRNA & nucleotide sequence in the coding
region of mRNA is then translated into amino acid sequence of protein)

• The genetic code (combination of 3 mRNA nucleotide)

- Codon AUG = start codon (methionine), codon GUU (valine), codon UAA/UAG/UGA = stop codon and
ribosome reads 1 codon at 1 time

. . . . . .

- Characteristic: degenerate but unambiguous ; universal (but exception occur at human mitochondrial
mRNA where UGA for tryptophan, AUA for methionine, CUA for threonine) ; non-overlapping (do not
contain extra-nucleotide or punctuation)

• Formation of aminoacyl-tRNA: amino acid attached to their tRNA by aminoacyl-tRNA synthehtase

(formation of ester bond) require energy.

Process of Translation
1. Initiation
• Formation of initiation complex composed of methionyl-tRNA , mRNA & ribosomes

- Met-tRNA initially form a complex with eukaryotic initiation factor 2 (elF) then bind to small
ribosomal subunit

- GTP on IF 2 is hydrolysed, IFs are released when large ribosomal subunit bind to form initiation
complex

• The ribosome is now complete, has 3 binding sites for tRNA: E (ejection), P (peptidyl) and A (aminosyl)

2. Elongation
• Binding of aminoacyl-tRNA to A site
aminosy(
- I EF direct binding of aminoacyl-tRNAIto coding mRNA in empty A site. GTP on EF hydrolysed (release
GDP and phosphate)

peptidy)
. . .

• Formation of peptide bond

- Peptidyltransferase catalyses peptide bond formation, transferring the initiation amino acid
L(methionine)3 from the P site onto amino acid at the A site

- -

• Translocation of the peptidyl-tRNA to P site

- Ribosomes moves a distances if 3 nucleotides along mRNA in 5’-3’ direction. What was in P site is
now in E site (uncharged tRNA) & what was in A site is now in P site (peptidyl tRNA) and A site is
empty for the next codon. This required another GTP and GTP on EF is hydrolyses

- Step is repeated until a termination/stop codon (UAG, UAA, UGA) enter the A site

3. Termination
• Stop codon is recognised by a release factor (RF) & altere the peptidyltransferase and add H2O instead
of amino acid

• Newly synthesised protein is release & ribosome break into 2 subunit

• GTP on RF is hydrolysed

Polysomes: each mRNA has multiple ribosomes

Processing of Protein: protein bind to nascent polypeptide chain and mediate the folding process by
mediator (chaperones: prevent improper interaction from occurring)

DNA translation inhibitor: streptomycin (prevent formation of initiation complex), tetracycline (inhibit
binding to A site), chloramphenicol (inhibit peptidyltransferase)

 15
REGULATION OF GENE EXPRESSION

Regulation of Prokaryotic
1. Transcription of mRNA from Bacterial operon

• The gene in operon are expressed coordinately either all turned on or all turned off

• If operon is expressed, all of its gene are transcribed. Product of transcription is single polycistronic
mRNA-multiple set of start/stop codon

2. Regulator of RNA pol binding by Role of Operator or by Repressor

• Inducer (eg: allolactose): bind to repressor, changing its conformation, inactive repressor no longer bind
to operator, RNA pol can bind to promoter region & transcription occur (bcs promotor activated)

• Corepressor (eg: tryptophan): bind to repressor, activating it, repressor-corepressor bind to operator &
prevent RNA pol from binding to promoter region & transcription cannot occur (bcs promotor repressed)

3. Lactose operon

• Lac operon ON (high lactose, low glucose)

- Lactose convert to allolactose (inducer), binds to repressor, causing its conformation changes that
prevent its from binding to operator. So operator is not blocked (for RNA pol)

- Low glucose, adenyl cyclase activate, cAMP high, cAMP bind to CRP form cAMP-CRP complex
causing RNA pol bind to promoter and initiate transcription

• Lac operon Off (low lactose, high glucose)

- No lactose: operator blocked by repressor molecule

- High glucose- adenyl cyclase inactivate, no cAMP, cAMP cannot bind to CRR, no cAMP-CRP
complex to initiate transcription

• Lac operon OFF (high lactose, high glucose)

- Even though repressor not bound to operator but bcs of present of glucose, no cAMP-CRP complex
to initiate RNA pol to bind to promotor so transcription cannot occur

4. Regulator of RNA pol binding by sigma factor

• σ factor (sigma) bind RNA pol to help RNA pol recognise promoter region on the DNA, RNA pol bind to
promoter then transcription can occur

5. Attenuation of transcription
• It trp is high/plentiful result in high level of Trp-tRNA & rapid translation of transcript. However rapid
translation generates hairpin loop in mRNA that serves as termination signal for RNA pol and
transcription stop

Regulation of Eukaryotic
DNA and chromosomes
1. Gene Rearrangment
• Segment of DNA can move from one location to another in the genome

• Eg: heavy chain gene from which lymphocytes produce Ig is generate by combining specific segment

2. Gene Amplification
• Certain region of chromosomes undergo repeated cycle of DNA replication

• Newly synthesised DNA is excised & forms small, unstable chromosomes called double minutes & this
will integrate into chromosomes thereby amplifying the gene in process

3. Gene Deletion
• Can occur through erros in DNA replication & cell division & are usually notice on if a disease result

Transcription
1. Gene specific regulatory proteins : TF, repressor, inducer, coactivator ad corepressor

2. Regulation of TF
• TF can be modulates by changes in amount of TF synthesis; binding of stimulatory/inhibitory ligand;
stimulation of nuclear entry

 16
Processing of transcript
1. Alternative splicing and polyadenylation sites
• Produce different protein from a same limited gene by use alternative splice sites (by addition of a cap to
5’-end; addition of poly (A) tail and removal of intron)

• Eg: production of membrane-bound antibody (IgM - intron already removed) & smaller secreted antibody
(IgD - intron not removed) form the same gene

2. RNA editing
• Ensure mature mRNA differs in different tissue

• Eg: Apo B made in liver & small intestine however in small intestine only conversion of C to U(through
deamination) in RNA transcript generate stop codon as as result protein produced in intestine (Apo B48)
short than in liver (Apo B100)

Initiation of translation & stability of mRNA

1. Initiation factor
• Translation usually involves the eIF which regulated through mechanism that involve phosphorylation

• Eg: heme regulate translation of global mRNA in reticulocytes by controlling phosphorylation of eIF

2. microRNA (miRNA)
• Regulate protein expression at post-transcriptional level, induced degradation of target mRNA or block
translation of target mRNA as result it will reduced expression of target mRNA

• °. α, β, γ , δ, Δ, ω, ε σ ρ
 16
Processing of transcript
1. Alternative splicing and polyadenylation sites
• Produce different protein from a same limited gene by use alternative splice sites (by addition of a cap to
5’-end; addition of poly (A) tail and removal of intron)

• Eg: production of membrane-bound antibody (IgM - intron already removed) & smaller secreted antibody
(IgD - intron not removed) form the same gene

2. RNA editing
• Ensure mature mRNA differs in different tissue

• Eg: Apo B made in liver & small intestine however in small intestine only conversion of C to U(through
deamination) in RNA transcript generate stop codon as as result protein produced in intestine (Apo B48)
short than in liver (Apo B100)

Initiation of translation & stability of mRNA

1. Initiation factor
• Translation usually involves the eIF which regulated through mechanism that involve phosphorylation

• Eg: heme regulate translation of global mRNA in reticulocytes by controlling phosphorylation of eIF

2. microRNA (miRNA)
• Regulate protein expression at post-transcriptional level, induced degradation of target mRNA or block
translation of target mRNA as result it will reduced expression of target mRNA

• °. α, β, γ , δ, Δ, ω, ε σ ρ