Biosynthesis of nucleotides

1. Biosynthesis of nucleotides Phar 6152 Spring 2004 Natalia Tretyakova, Ph.D. Required reading: Stryer’s Biochemistry 5th edition, p. 262-268, 693-712 (or Stryer’s Biochemistry 4th edition p. 238-244, 739-759)

2. Tentative Lecture plan: Biosynthesis of Nucleotides 03-31 Introduction. Biological functions and sources of nucleotides. Nucleotide metabolism. 04-02 Biosynthesis of pyrimidine ribonucleotides. 04-05 Biosynthesis of purine ribonucleotides 04-07 Biosynthesis of deoxyribonucleotides. Inhibitors of nucleotide metabolism as drugs. 04-09 Review 04-12 Exam

3. Biological functions and sources of nucleotides. Nucleotide metabolism Required reading: Stryer’s Biochemistry 5th Ed., p. 693-694, 709-711

4. Biological functions of nucleotides 1. Building blocks of nucleic acids (DNA and RNA). 2. Involved in energy storage, muscle contraction, active transport, maintenance of ion gradients. 3.      Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine). 4.      Components of coenzymes (NAD+, NADP+, FAD, FMN, and CoA) 5.      Metabolic regulators: a.      Second messengers (cAMP, cGMP) b.      Phosphate donors in signal transduction (ATP) c.       Regulation of some enzymes via adenylation and uridylylation

5. Nucleotides b-glycosidic bond RNA- ribose (R) DNA – deoxyribose (dR)

6. Nucleobase structures

7. Hypoxanthine Inosine Inosinate (IMP) Xanthine Xanthosine Xanthylate (XMP)

8. Two major routes for nucleotide biosynthesis dNTPs dNTPs Stryer Fig. 25.1

9. Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via salvage pathways 1 and 2 (red) 1 2

10. Phosphoribosyl transferases involved in salvage pathway convert free bases to nucleotides a d e n i n e p h o s p h o r i b o s y l t r a n s f e r a s e A d e n y l a t e + P P i A d e n i n e + P R P P h y p o x a n t h i n e - g u a n i n e (HGPRT) p h o s p h o r i b o s y l t r a n s f e r a s e G u a n y l a t e + P P i G u a n i n e + P R P P Inosinate H y p o x a n t h i n e + P R P P + P P i

11. Biodegradation of Nucleotides (Stryer p. 709-711)

12. Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via salvage pathways 1 and 2 (red) 1 2

13. Purine biodegradation in humans leads to uric acid

14. AMP is deaminated to IMP AMP deaminase

15. IMP is deribosylated to hypoxanthine phosphorylase

16. Hypoxanthine is oxidized to xanthine

17. Guanine can be deaminated to give xanthine

18. Uric acid is the final product of purine degradation in mammals

19. Uric acid is excreted as urate

20. Deleterious consequences of defective purine metabolism • Gout (excess accumulation of uric acid) • Lesch-Nyhan syndrome (HGPRT null) • Immunodeficiency

21. Gout • Precipitation and deposition of uric acid causes arthritic pain and kidney stones • Causes: impaired excretion of uric acid and deficiencies in HGPRT

22. Lesch-Nyhan Syndrome • Caused by a severe deficiency in HGPRT activity • Symptoms are gouty arthritis due to uric acid accumulation and severe neurological malfunctions including mental retardation, aggressiveness, and self-mutilation • Sex-linked trait occurring mostly in males

23. Lack of HGPRT activity in Lesch-Nyhan Syndrome causes a buildup of PRPP, which activates the synthesis of purine nucleotides h y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e G u a n y l a t e + P P i G u a n i n e + P R P P H y p o x a n t h i n e + P R P P I nosinate + P P i • Excessive uric acid forms as a degradation product of purine nucleotides • Basis of neurological aberrations is unknown

24. Immunodeficiency induced by Adenosine Deaminase defects AMP deaminase • Defects in AMP deaminase prevent biodegradation of AMP • AMP is converted into dATP • dATP inhibits the synthesis of deoxyribonucleotides by ribonucleotide reductase, causing problems with the immune system (death of lymphocytes, immunodeficiency disease)

25. Summary: • Nucleotides have many important functions in a cell. • Two major sources of nucleotides are salvage pathway and de novo biosynthesis • Purine nucleotides are biodegraded by nucleotidases, • nucleotide phosphorylases, deaminases, and • xanthine oxidase. • Uric acid is the final product of purine biodegradation in mammals • Defective purine metabolism leads to clinical • disease.

26. Key concepts in Biosynthesis: Review • Committed step • Regulated step • Allosteric inhibitor • Feedback inhibition

27. De novo Biosynthesis of Pyrimidines Required reading: Stryer’s Biochemistry 5th Ed., p. 262-267, 694-698

28. De novo Biosynthesis of Pyrimidines dTTP Stryer Fig. 25.2

29. Part 1. The formation of carbamoyl phosphate Enzyme: carbamoyl phosphate synthetase II (CPS) This is the regulated step in pyrimidine biosynthesis

30. Bicarbonate is phosphorylated CPS

31. Phosphate is displaced by ammonia: : CPS General strategy for making C-N bonds: C-OH is phosphorylated to generate a good leaving group (phosphate)

32. General Mechanism for making C-N bonds:

33. Ammonia necessary for the formation of carbamic acid originates from glutamine:

34. Structure of Carbamoyl phosphate synthetase II Stryer Fig. 25.3

35. The active site for glutamine hydrolysis to ammonia contains a catalytic dyad of Cys and His residues Stryer Fig. 25.4

36. Carbamic acid is phosphorylated CPS

37. Substrate channeling in CPS Stryer Fig. 25.5

38. Carbamoyl phosphate supplies the C-2 and the N-3 of the pyrimidine ring dTTP

39. Part 2. The formation of orotate.

40. Aspartate is coupled to carbamoyl phosphate Enzyme: aspartate transcarbamoylase This is the committed step in pyrimidine biosynthesis

41. Aspartate transcarbamoylase is allosterically inhibited by CTP Stryer Fig. 10.2

42. Allosteric regulation of Aspartate Transcarbamoylase Stryer Fig. 10.5

43. PALA is a bisubstrate analog that mimics the reaction intermediate on the way to carbamoyl aspartate Bisubstrate analog

44. PALA binds to the active site within catalytic subunit Stryer Fig. 10.7

45. Substrate binding to Aspartate Transcabamoylase induces a large change in ATC quaternary structure Stryer Fig. 10.8

46. CTP binding prevents ATC transition to the active R state Stryer Fig. 10.9

47. Allosteric regulation of Aspartate Transcabamoylase Stryer Fig. 10.10

48. N-Carbamoylaspartate cyclizes to dihydroorotate - H2O

49. Dihydroorotate is oxidized to orotate Dihydroorotate dehydrogenase

50. Part 3. The formation of UMP a. Orotate is phosphoribosylated to OMP Pyrimidine phosphoribosyl transferase