Pyrimidine Nucleotide synthesis

Question 1

Pyrimidines and difference in synthesis

Answer 1

from aspartic acid, amide nitrogen from glutamine, Co2

purine was built on the sugar, py ring is first made, then converted into a nucleotide
-fewer steps and fewer ATP

Question 2

CPS II def, differences to I

Answer 2

Carbomoyl Phosphate II high energy compound required for the syn of both py and urea

carbomoyl phosphate required for py and urea sun in formed by two isoenzymes I and II

py: (CPSII in cytosol)
urea: CPS I in mito

look at comparison chart on slide 8

FXN

• first committed step in py synth
• PRPP (also for pur) and ATP pos allosteric effectors
• ATP provides coordination between both pur and py
• UTP negative allosteric effector

Question 3

first step in py syn

Answer 3

2ATP+CO2+glutamine-CPSII-> carbomoyl phosphate
-kicks out glutamate and 2 ADP

multifunctional protiens take over, increase speed and efficiency

orotic acid: first py base formed

OMP-first pyrimidine nucleotide formed
converted to UMP

UMP converted to UDP (nucleoside monophosphate kinase)

UMP converted to UTP (broad nucleoside diphosphate kinase)

Question 4

CTP formation

Answer 4

second pyrimidine nucleotide made by transferring the amide group from glutamine to UTP in a reaction catalyzed by CTP SYNTHASE (ATP needed)

activated by UTP
Inhibited by CTP

Question 5

conversion of ribonucleotides to deoxyribonucleotides

Answer 5

nucleotides need to be reduced to 2’ deoxyribose
RIBONUCLEOTIDE REDUCTASE
-rate limiting step
-TDP is NOT a substrate
-requires peptide coenzyme THIOREDOXIN which donates electornes used to reduce the hydroxyl group
-thioredoxin congtains a disulfide which is reduced a second time to THIOREDOXIN REDUCTASE. NADPH is e donor

ADP-dADP
GDP-dGDP
CDP-dCDP
UDP-cUDP

-can only use diphosphates and end up with diphosphates

slide 16 Question

Question 6

regulation of RNR

Answer 6

unusual bc it occurs at its regulatory site and substrate specificity site
1. active site-binding of the substrate ADP, GDP, CDP, UDP

2. regulatory site
   -ATP is pos allosteric inhibitor
   dATp is an inhibitor of the entire enzyme
3. substrate specificity siteL when dTTP is bout at this site, GDP is the substrate but not CDP

Why is RNR so finely tuned: proper intracellular ratios are essential, deficiency of one is lethal and excess is mutagenic due to erroneous incorporation

Question 7

Adenosine Deaminase

Answer 7

deoxy Adenosine to deoxyinosine….eventually to uric acid

witout ADA-increase in dAdenosine, increase in dATP, RNR activity decrease, dNTP synthesis decrease, DNA replication stalled

ADA leades to severe combined immunodeficiency desiease

human lymphocytes have the highest levels of ADA

patients lack bothe T and B cells

Bubble baby
gene therapy

Question 8

Thmymidylate Synthase

Answer 8

catalyzes the formation of thymidine monophosphate (dTMP) from dUMP

thymidine NEVER exists with a ribose

-rate limiting step for DNA synthesis!

dUTPase: dUTP-> dUMP +ppi
dCMP deaminase: dCMP-> dUMP + nH3

THF required for synthesis of all purines (AMP GMP) and thymidine monophosphate dTMP but not for synthesis of UTP or CTP

need methylene hydrofolate, which donates a methyl group and two electors (tetrahydrofolate is oxidized to dihydrofolate (folic acid))
DIHYDROFOLATE REDUCTASE reconverts DHF back to THF with NADPH

folate def, results in decreased production of dTMP and therefore an increased incorporation of dUTP into DNA

Question 9

why use thymidine nucleoties and not uracil nucleotides

Answer 9

cytosine is unstable and undergoes spontaneous deamination to form uracil. if uracil were in DNA, DNA repair would not know where there was a deamination event and just a normal uracil

Question 10

Salvage of pyrimidine

Answer 10

not as sig as purines
-only 30% salvaged

requires ATP

occurs as nucleosides rather than free bases
-can do bases too but very slow

-py nucleosides travel in blood and are taken up by tissues

kinases add Phosphates back on

Question 11

degradation of py nucleotides

Answer 11

products of py are highly water soluble, unlike purines

1. py nucleotides dephosphorylated and cleaved to produce ribose 1 phosphate and the free py bases cytosine uracil and thymine
2. bases converted to Co2 and NH4+ excreted in urine or converted to urea
   - no problems fo the body, no uric acid
   - no energy produced

Question 12

Digestion of dietary nucleotides

Answer 12

degredation of nucleic acids occus in SI by panc enzymes, to free bases and nucleosides

only small amount 5% ingested are absorbed

dietary pur and py gen converted to degradation products by intestinal mucosa cells and excreted in urine

Question 13

Chemotherapy analogues (5)

Answer 13

Rapidly growing cancers have greater requirements for DNA and RNA precursosr
-generally more sensitive than normal cells to inhibition of nucleotide biosyn

thymidine nucleotide biosyn is a target for cancer therapy , bc its ONLY used in DNA syn.

most normal cells require less dTMP less sens to agents that inh thymidylate synthase or dihydrofolate reductase

Methotrexate
-comp inhibitor of dihydrofolate reductaseL no thymidylate synthase

TrimethoprimL inhibit bacterial dihydrofolate reductase, antibacterial agent (bac enzyme stonger affinity for molecule)

5-Flourouracil suicide inhibitor
-admined as a bse, cells convert it to F-dUMP-irreversible suicide inhibitor, DNA rep stopped.

hydroxyurea: antineoplastic drug inhibits ribonucleotide reductase. RNR
dNTPs production decreases, terat sicke cell

Azidothymidine (AZT) nucleoside that contains azido group N3 on 3’ deoxyribose
-inhibits DNA rep when converted to AZT monophosphate and triphosphate, competes with dTTP