Nucleotide metabolism – Part 1 (purine biosynthesis)

1. Nucleotide metabolism – Part 1(purine biosynthesis) By Henry Wormser, Ph.D Professor of Medicinal Chemistry

2. Biological significance of nucleotide metabolism • Nucleotides make up nucleic acids (DNA and RNA) • Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP) • Many metabolic pathways are regulated by the level of the individual nucleotides • Example: cAMP regulation of glucose release • Adenine nucleotides are components of many of the coenzymes • Examples: NAD+, NADP+, FAD, FMN, coenzyme A

3. Dietary nucleotides • do not contribute energy as do carbs, proteins and fats • are not incorporated into RNA or DNA unless given I.V. • normally metabolized to individual components (bases, sugar and phosphate) • purines are converted to uric acid which is then excreted

4. Medical significance of nucleotide metabolism • Anticancer agents: • Rapidly dividing cells biosynthesize lots of purines and pyrimidines, but other cells reuse them. Cancer cells are rapidly dividing, so inhibitor of nucleotide metabolism kill them • Antiviral agents • Zidovudine (Retrovir) • Lamivudine (Epivir) • Valacyclovir (Valtrex)

6. Structures of nucleotide building blocks and nucleotides

7. Structures of nucleotide buildingblocks and nucleotides guanine: comes from guano; thymine –thymus gland

8. Ribonucleotide – phosphate = ribonucleoside

9. Biosynthesis of the purine nucleotide system

10. Synthesis of Inosine Monophosphate • Basic pathway for biosynthesis of purine ribonucleotides • Starts from ribose-5-phosphate which is derived from the pentose phosphate pathway • Requires 11 steps overall • occurs primarily in the liver

11. The big picture

12. Steps 1 thru 3 • Step 1:Activation of ribose-5-phosphate • enzyme: ribose phosphate pyrophosphokinase • product: 5-phosphoribosyl-a-pyrophosphate (PRPP) • PRPP is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine and tryptophan

13. Step 1: purine synthesis

14. Steps 1 thru 3 • Step 2: acquisition of purine atom 9 • enzyme: amidophosphoribosyl transferase • displacement of pyrophosphate group by glutamine amide nitrogen (inversion of configuration – a to b • product: b-5-phosphoribosylamine Steps 1 and 2 are tightly regulated by feedback inhibition

15. Step 2: purine synthesis:commited step

16. Steps 1 thru 3 • Step 3: acquisition of purine atoms C4, C5, and N7 • enzyme: glycinamide synthetase • b-phosphoribosylamine reacts with ATP and glycine • product: glycinamide ribotide (GAR)

17. Step 3 : purine synthesis

18. Steps 4 thru 6 • Step 4: acquisition of purine atom C8 • formylation of free a-amino group of GAR • enzyme: GAR transformylase • co-factor of enzyme is N10-formyl THF • Step 5: acquisition of purine atom N3 • The amide amino group of a second glutamine is transferred to form formylglycinamidine ribotide (FGAM) • Step 6: closing of the imidazole ring or formation of 5-aminoimidazole ribotide

20. Step 6: purine synthesis

21. Step 7 • Step 7: acquisition of C6 • C6 is introduced as HCO3- • enzyme: AIR carboxylase (aminoimidazole ribotide carboxylase) • product: CAIR (carboxyaminoimidazole ribotide) • enzyme composed of 2 proteins: PurE and PurK (synergistic proteins)

22. Step 7: purine synthesis

23. Steps 8 thru 11 • Step 8: acquisition of N1 • N1 is acquired from aspartate in an amide condensation reaction • enzyme: SAICAR synthetase • product: 5-aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR) • reaction is driven by hydrolysis of ATP

24. Step 8: purine synthesis

25. Steps 8 thru 11 • Step 9: elimination of fumarate • Enzyme: adenylosuccinate lyase • Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR) • Step 10: acquisition of C2 • Another formylation reaction catalyzed by AICAR transformylase • Product: 5-formaminoimidazole-4-carboxamide ribotide (FAICAR)

26. Step 9: purine synthesis

27. Step 10: purine synthesis

28. Step 11 • cyclization or ring closure to form IMP • water is eliminated • in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis • once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells

29. Step 11: purine synthesis

31. Synthesis of adenine and guanine nucleotides

32. Purine nucleoside diphosphates and triphosphates: - to be incorporated into DNA and RNA, nucleoside monophosphates (NMP’s) must be converted into nucleoside triphosphates (NTP’s) - nucleoside monophosphate kinases (adenylate & guanylate kinases) - nucleoside diphosphate kinase

33. Regulation of purine nucleotide biosynthesis

35. The purine salvage pathway • Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides • Two phosphoribosyl transferases are involved: • APRT (adenine phosphoribosyl transferase) for adenine • HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine

36. Salvage of purines Adenine phosphoribosyltransferase (APRT)

37. Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in neural tissue) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Hypoxanthine + PRPP IMP + Ppi Guanine + PRPP GMP + Ppi Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cells Salvage of purines

38. Lesch-Nyhan syndrome • there is a defect or lack in the HGPRT enzyme • the rate of purine synthesis is increased about 200X • uric acid level rises and there is gout • in addition there are mental aberrations • patients will self-mutilate by biting lips and fingers off

39. Lesch-Nyhan syndrome

40. Salvage of purine bases

41. Next: Part 2 - biosynthesis of pyrimidine nucleotides

42. Nucleotide metabolism – Part 2(pyrimidine biosynthesis) By Henry Wormser, Ph.D Professor of Medicinal Chemistry

43. Synthesis of pyrimidine ribonucleotides • shorter pathway than for purines • base is made first, then attached to ribose-P (unlike purine biosynthesis) • only 2 precursors (aspartate and glutamine, plus HCO3-) contribute to the 6-membered ring • requires 6 steps (instead of 11 for purine) • the product is UMP (uridine monophosphate)

44. Origin of atoms in pyrimidine ring

45. The big picture

46. Step 1: synthesis of carbamoyl phosphate • Condensation of glutamine, bicarbonate in the presence of ATP • Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme used here

47. Step 1: pyrimidine synthesis CPS-II is the major site of regulation in animals: UDP and UTP inhibit the enzyme and ATP and PRPP activate it It is the committed step in animals

48. Step 2: synthesis of carbamoyl aspartate • enzyme is aspartate transcarbamoylase (ATCase) • catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Pi • ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTP • carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step

49. Step 2: pyrimidine synthesis

50. Step 3: ring closure to form dihydroorotate • enzyme: dihydroorotase • forms a pyrimidine from carbamoyl aspartate • water is released in this process