1. Nucleic Acid Metabolism Robert 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 Base Nucleoside Nucleotide Ribose
Deoxyribose
Base
Purines
Pyrimidines
Nucleoside
Base plus sugar
Nucleotide
E.g., AMP, ADP, ATP

3. Nomenclature DNA Purine Bases Purine Nucleosides
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) Utilizes (Substrates)
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