Nucleotide Metabolism C483 Spring 2013

1. A ribose sugar is added to ________ rings after their synthesis and to ________ rings during their synthesis. A) purine; pyrimidine B) pyrimidine; purine C) purine; purine D) pyrimidine; pyrimidine 2. The first nucleotide product in the de novo biosynthetic pathway of purines is A) AMP. B) GMP. C) IMP. D) XMP. 3. Which of the following statements is false concerning purine synthesis? A)N7 is from glycine B)C2 is from carbon dioxide (bicarbonate) C)N3 is from glutamine D)C8 is from 10-formylTHF.

4. Which is a precursor in the de novo synthesize CTP? A) CMP. B) GMP. C) TMP. D) UMP. 5.Which of the following is not a role of a catalytic sulfur atom in ribonucleotide reductase? A)Proton donor B)Radical stabilization C)Redox reaction D)Covalent catalysis 6. Dihydrofolate reductase and thymidylate synthetase are major targets for anticancer drugs because A) these enzymes are unique in cancer cells. B) cancer cells lack sufficient amounts of these enzymes. C) cancer cells grow rapidly and are very dependent upon the activities of these enzymes. D) they donate one-carbon groups. E) All of the above.

Terminology of Nucleic Acids Nucleotide Nucleoside Nucleobase AMP ADP ATP dAMP

Some Examples of Nucleotides ATP GTP S-AM FAD NAD + UDP-Glucose CoA

De Novo Synthesis

De Novo Synthesis of Purines Form activated ribose Form 5-phospho ribosylamine Build IMP from precursors Synthesis of AMP and GMP

PRPP Pentose phosphate pathway 2 ATP equivalents Over production of PRPP is one cause of gout because PRPP stimulates the next step…

5-phosphoribosylamine First step of purine biosynthesis Glutamine is N donor Regulated – Activation by PPRP – Increased purine levels – Degradation of purines leads to compound which can cause gout

Purine Pathway Don’t need to know details, order Know precursors – N from Asp, Gln – C from THF, Gly, CO2 Cost – 2 ATP eq for PRPP – 5 more ATP steps Know this figure!

Purines Two distinct strategies for amination – Mechanisms Regulation – Feedback to 5-phospho ribosylamine – Branchpoint regulation

Compare/Contrast Purine biosynthesis – Salvage is a major pathway – Base synthesized while attached to ribose – IMP is common intermediate for AMP and GMP, but itself is not a typical nucleotide Pyrimidine biosynthesis – De novo is a major pathway – Base is synthesized, then attached to ribose – UMP, a typical nucleic acid, is converted into other pyrimidines

De novo Pyrimidine Synthesis First step regulated (compare to urea cycle) Asp is different than purine— whole molecule is incorporated

Further Modifications Interconversion of nucleotides (mono, di, tri phosphates) Reduction to form deoxynucleotides Methylation to form dTMP

Nucleotide Interconversions Fast, reversible, driven by high [ATP] NMP  NDP catalyzed by specific nucleoside monophosphate kinase NDP  NTP catalyzed by nonspecific kinase AMP + ATP  ADP + ADP important in energy balance

Deoxyribonucleotides Deoxygenation occurs on diphosphates One enzyme affects all transformations [dUDP]

Ribonucleotide Reductase Sulfur does amazing chemistry! – Stable radical – Proton donor – Redox reagent NADPH is ultimate source of reducing

Regulation of Reductase One enzyme balances needs of cell via regulation of activity and selectivity Be able to explain why this table makes sense

Methylation dTMP is made from dUMP Key step in replicating cells Therapeutic target for anti-cancer drugs Two key enzymes

Thymidylate Synthase Methylene-THF acts as a “methyl” donor – Donates methylene – And hydride Fascinating chemistry! – Sulfur is covalent catalyst – Internal 1,3-hydride shift THF is left as DHF

5-Fluorouracil Incorporated into monophosphate nucleotide in body Mechanism based inhibitor (Trojan Horse) Forms covalent link to enzyme like normal No elimination possible because proton replaced with fluorine

DHF reductase DHF must be reduced back to THF to be a viable cofactor Second chemotherapy target Competitive inhibitor that is structurally similar to THF would end methylation process

Review of Purines Knowing blue in figure will help with chapter summary

Review of Pyrimidines Knowing blue in figure will help with chapter summary

Catabolism Less important than other catabolic processes – not a major energy source – Lots of salvage – Serves to clear excess In humans, purines  uric acid (excreted) In humans, pyrimidines  acetyl CoA, succinyl CoA for some energy gain

Severe Combined Immunodeficiency Syndrome (SCIDS) Deficiency of adenosine deaminase First step in catabolism High levels of dATP lead to low levels of dNTP No DNA kills fast growing T-cells *

Answers 1.B 2.C 3.B 4.D 5.D 6.C