Nucleic Acid Metabolism Robert F. Waters, PhD

1. Nucleic Acid MetabolismRobert F. Waters, PhD • Nucleotides • Essential for all cells • Carriers of activated intermediates in carbohydrate, lipids and proteins • CoA • FAD • NAD • NADP • Energy Carriers • ATP • Inhibiting or activating enzymes • DNA • RNA

2. Nucleotide Structure • Ribose Sugar • Ribose • Deoxyribose • Base • Purines • Pyrimidines • Nucleoside • Base plus sugar • Nucleotide • E.g., AMP, ADP, ATP

3. Nomenclature • DNA Purine Bases • Adenine • Guanine • Purine Nucleosides • Adenosine • Guanosine • DNA Nucleotides (Purine) • dAMP (deoxyadenylate) • dGMP (deoxyguanylate) • RNA Nucleotides (Purine) • Adenylate (AMP) • Guanylate (GMP)

4. Nomenclature Continued • DNA Pyrimidine Bases • Thymine • Cytosine (Also RNA) • DNA Pyrimidine Nucelosides • Thymidine • Cytidine • DNA Pyrimidine Nucleotides • (dTMP) deoxythymidylate • (dCMP) deoxycytidylate • RNA Pyrimidine Nucleotides • (CMP) cytidylate • (UMP) uridylate

5. PRPP 5-Phosphoribosyl 1-Pyrophosphate • Addition of the ribose sugar component • HMP • ATP Required • Mg++ • Pi activates and nucleosides inhibit

6. Pyrimidine Synthesis • UMP (Uridine 5-monophosphate) to UTP • Precursor to CTP • Occurs on mitochondria inner membrane • Carbamoyl phosphate synthetase II • Different from CPS I • CPS I uses free ammonia • CPS II uses glutamine for amino source

7. Carbamoyl Phosphate Synthetase II

8. Formation of Uridine 5’-phosphate

9. Enzymes of Pyrimidine Biosynthesis

10. UTP to CTP Conversion • CTP Synthetase Reaction

11. Conversion of Ribonucleotides to Deoxyribonucleotides • Ribonucleotide reductase • NADP • Thioredoxin reductase • Example is production of dCDP

12. Allosteric Inhibition of Ribonucleotide Reductase • ATP activates • dATP inhibits

13. Thymidylate Biosynthesis • Substrates and Vitamins • dUMP • Folate (N5, N10,-Methylene-THF) • Glycine/Serine • NADP

14. Conversion of dUMP to dTMP:Overall • 5-fluorouracil • Methotrexate

15. Thymidylate Pathway:Specific

16. Thymidylate Synthesis and Cancer Chemotherapy • Thymidylate synthase is target for fluorouracil • Action is 5-fluorouracil (5-FU)is converted to 5-fluoro-2’-deoxyuridylate (dUMP structural analog) • Then 5-fluoro-2’-deoxyuridylate binds to the enzyme Thymidylate Synthase and undergoes a partial reaction where part of the way through 5-fluoro-2’-deoxyuridylate forms a covalent bridge between Thymidylate Synthase and N5, N10-Methylene THF and is an irreversible inhibition. • Normally, the enzyme, Thymidylate Synthase and the vitamin would NOT be linked together permanently • This type of inhibition is called “suicide-based enzyme inhibition” because the inhibitor participates in the reaction causing the enzyme to react with the compound producing a compound that inactivates the enzyme itself.

17. Fluorouracil Pathway Suicide inhibition because Flurouracil does not directly inhibit enzyme.

18. Methotrexate • Competitive inhibitor of Dihydrofolate Reductase • Used in, • Acute lymphoblastic leukemia • Osteosarcoma in children • Solid tumor treatment • Breast, head, neck, ovary, and bladder • Prevents regeneration of tetrahydrofolate and removes activity of the active forms of folate

19. Leucovorin Rescue Strategy in Methotrexate Chemotherapy • Patients given sufficient methotrexate that if were not followed by Leucovorin (N5-methenyl-THF) would be fatal. • All neoplastic cells are killed • Patients are “rescued” (6-36 hours) by the Leucovorin (Folate) otherwise would die due to permanent tetrahydrofolate shutdown. • Tumor resistance to methotrexate can occur in patients who have “gene amplification” of dihydrofolate reductase (in tumor cells) • More dihydrofolate reductase is produced by more than the normal active genes usually present in normal cells.

20. Purine Biosynthesis • IMP (Inosine Monophosphate) • Precursor to • GMP and AMP • Utilizes (Substrates) • Glycine • Glutamine • ATP • Folate (N10-formyl-THF) • Aspartate • CO2 • PRPP amidotransferase is rate limiting • Inhibited by AMP and GMP

21. IMP Pathway

22. IMP to AMP and GMP • Glutamine, NAD, ATP used in GMP production • Aspartate, GTP used AMP production

23. AMP and GMP Pathway

24. Nucleotide Pyrimidine Catabolism • Degradation of pyrimidine metabolites • UMP, CMP, TMP • End products are acetyl-CoA and Propionyl-CoA • Ribose sugar component may be converted to ribose-5-phosphate which is a substrate for PRPP Synthetase • Ribose sugar component may be further catabolized in HMP pathway

25. Pyrimidine Catabolic Pathway

26. Purine Catabolism

27. Regulation of Nucleotide Metabolism • Pyrimidine Regulation • Primary regulatory step is Carbamoyl Phosphate via Carbamoyl Phosphate Synthetase II • Purine Regulation

28. Action of Allopurinol • Allopurinol is purine base analog • Three mechanisms • Allopurinol is oxidized to alloxanthine by xanthine dehydrogenase • Then Allopurinol and alloxanthine are inhibitors of xanthine dehydrogenase • This inhibition decreases urate formation • Then concentrations of Allopurinol and alloxanthine increase but do not precipitate as urate does. • Allopurinol and alloxanthine are excreted into the urine

29. Action of Allopurinol:Pathway

30. Biosythesis of Nucleotide Coenzymes • CoA • OTC is pantothenate • Uses ATP, CTP, Cysteine

31. Coenzyme A Pathway

32. FMN and FAD • OTC is riboflavin • Consumes ATP