Nucleotides : Synthesis and Degradation

1. Nucleotides: Synthesis and Degradation

2. Roles of Nucleotides • Precursors to nucleic acids (genetic material and non-protein • enzymes). • Currency in energy metabolism (eg. ATP, GTP). • Carriers of activated metabolites for biosynthesis • (eg. CDP, UDP). • Structural moieties of coenzymes (eg. NAD, CoA). • Metabolic regulators and signal molecules (eg. cAMP, • cGMP, ppGpp).

3. Biosynthetic routes: De novo and salvage pathways De novo pathways Almost all cell types have the ability to synthesize purine and pyrimidine nucleotides from low molecular weight precursors in amounts sufficient for their own needs. The de novo pathways are almost identical in all organisms. Salvage pathways Most organisms have the ability to synthesize nucleotides from nucleosides or bases that become available through the diet or from degredation of nucleic acids. In animals, the extracellular hydrolysis of ingested nucleic acids represents the major route by which bases become available.

4. Reutilization and catabolism of purine and pyrimidine bases blue-catabolism red-salvage pathways endonucleases: pancreatic RNAse pancreatic DNAse phosphodiesterases: usually non-specific

5. PRPP: a central metabolite in de novo and salvage pathways PRPP synthetase Enzyme inhinited by AMP, ADP, and GDP. In E. coli, expression is repressed by PurR repressor bound to either guanine or hypoxanthine. Roles of PRPP: his and trp biosynthesis, nucleobase salvage pathways, de novo synthesis of nucleotides

6. Purine Nucleotide Synthesis

7. Example of a salvage pathway: guanine phosphoribosyl transferase In vivo, the reaction is driven to the right by the action of pyrophosphatase Shown: HGPRT, cells also have a APRT.

8. De novo biosynthesis of purines: low molecular weight precursors of the purine ring atoms

9. Synthesis of IMP The base in IMP is called hypoxanthine Note: purine ring built up at nucleotide level. precursors: glutamine (twice) glycine N10-formyl-THF (twice) HCO3 aspartate In vertebrates, 2,3,5 catalyzed by trifunctional enzyme, 6,7 catalyzed by bifunctional enzyme.

10. Pathways from IMP to AMP and GMP G-1: IMP dehydrogenase G-2: XMP aminase A-1: adenylosuccinate synthetase A-2: adenylosuccinate lyase Note: GTP used to make AMP, ATP used to make GMP. Also, feedback inhibition by AMP and GMP.

11. Pathways from AMP and GMP to ATP and GTP Conversion to diphosphate involves specific kinases: GMP + ATP <-------> GDP + ADP Guanylate kinase AMP + ATP <-------> 2 ADP Adenylate kinase Conversion to triphosphate by Nucleoside diphosphate kinase (NDK): GDP + ATP <------> GTP + ADP DG0’= 0 ping pong reaction mechanism with phospho-his intermediate. NDK also works with pyrimidine nucleotides and is driven by mass action.

12. Allosteric regulation of purine de novo synthesis

13. Purine degredation AMP deamination in muscle, hydrolysis in other tissues. Xanthine oxidase:contains FAD, molybdenum, and non-heme iron. In primates, uric acid is the end product, which is excreted.

14. Purine degredation in other animals

15. Clinical disorders of purine metabolism Excessive accumulation of uric acid: Gout The three defects shown each result in elevated de novo purine biosynthesis

16. Common treatment for gout: allopurinol Allopurinol is an analogue of hypoxanthine that strongly inhibits xanthine oxidase. Xanthine and hypoxanthine, which are soluble, are accumulated and excreted.

17. Diseases of purine metabolism (continued) Lesch-Nyhan Syndrome: Severe HGPRT deficiency In addition to symptoms of gout, patients display severe behavioral disorders, learning disorder, aggressiveness and hostility, including self-directed. Patients must be restrained to prevent self-mutilation. Reason for the behavioral disorder is unknown. X-linked trait (HGPRT gene is on X chromosome). Severe combined immune deficiency (SCID): lack of adenosine deaminase (ADA). Lack of ADA causes accumulation of deoxyadenosine. Immune cells, which have potent salvage pathways, accumulate dATP, which blocks production of other dNTPs by its action on ribonucleotide reductase. Immune cells can’t replicate their DNA, and thus can’t mount an immune response.

18. De novo pyrimidine biosynthesis Pyrimidine ring is assembled as the free base, orotic acid, which is them converted to the nucleotide orotidine monophosphate (OMP). The pathway is unbranched. UTP is a substrate for formation of CTP.

19. Pyrimidine Synthesis

20. De novo synthesis of pyrimidines 1: carbamyl phosphate synthase 2: aspartate transcarbamylase 3: dihydroorotase 4: dihydroorotate DH 5: orotate phosphoribosyl tranferase 6: orotidylate decarboxylase 7: UMP kinase 8: NDK 9: CTP synthetase CAD=1,2,3 5 +6=single protein

21. Regulation of pyrimidine de novo synthesis

22. Catabolism of pyrimidines

23. Overview of dNTP biosynthesis One enzyme, ribonucleotide reductase, reduces all four ribonucleotides to their deoxyribo derivitives. A free radical mechanism is involved in the ribonucleotide reductase reaction. There are three classes of ribonucleotide reductase enzymes in nature: Class I: tyrosine radical, uses NDP Class II: adenosylcobalamin. uses NTPs (cyanobacteria, some bacteria, Euglena). Class III: SAM and Fe-S to generate radical, uses NTPs. (anaerobes and fac. anaerobes).

24. Structure of rNDP reductase (E. coli, ClassI)

25. Proposed mechanism for rNDP reductase

26. Proposed reaction mechanism for ribonucleotide reductase

27. Sources of reducing power for rNDP reductase

28. Biological activities of thioredoxin

29. Regulation of activities of mammalian rNDP reductase

30. Salvage and de novo pathways to thymine nucleotides

31. Substrate recvognition by dUTPase

32. Relationship between thymidylate synthase and enzymes of tetrahydrofolate metabolism

33. Catalytic mechanism of thymidylate synthase

34. Regeneration of N5, N10-methylenetetrahydrofolate

35. Biosynthesis of NAD+ and NADP+

36. Biosynthesis of CoA from pantothenate

37. Proposed reaction mechanism for FGAM synthetase

38. The transformylation reactions are catalyzed by a multiprotein complex components of the complex: GAR transformylase (3) AICAR transformylase (9) serine hydroxymethyl transferase, trifunctional formylmethenyl-methylene-THF synthase (activities shown with asterisk)

39. Proposed catalytic mechanism for OMP decarboxylase

40. Reactions catalyzed by eukaryotic dihydroorotate dehydrogenase

41. Nitrogenous Bases • Planar, aromatic, and heterocyclic • Derived from purine or pyrimidine • Numbering of bases is “unprimed”

42. Nitrogenous Bases Pyrimidines Purines N1: Aspartate Amine C2, C8: Formate N3, N9: Glutamine C4, C5, N7: Glycine C6: Bicarbonate Ion

43. Nucleotide Metabolism • PURINE RIBONUCLEOTIDES: formed de novo • i.e., purines are not initially synthesized as free bases • First purine derivative formed is Inosine Mono-phosphate (IMP) • The purine base is hypoxanthine • AMP and GMP are formed from IMP

44. Purine Nucleotides • Get broken down into Uric Acid (a purine)

45. Purine Nucleotide Synthesis • ATP is involved in 6 steps and an additional ATP is needed to form the first molecule (R5P) • PRPP in the first step of Purine synthesis is also a precursor for Pyrimidine Synthesis, His and Trp synthesis • Role of ATP in first step is unique– group transfer rather than coupling • In second step, C1 notation changes from a to b (anomers specifying OH positioning on C1 with respect to C4 group) • In step 3, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)

46. Coupling of Reactions • Hydrolyzing a phosphate from ATP is relatively easy G°’= -30.5 kJ/mol • If endergonic reaction released energy into cell as heat energy, wouldn’t be useful • Must be coupled to an exergonic reaction • When ATP is a reactant: • Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl, or adenosinyl group in transferase reaction OR • ATP hydrolysis can drive an otherwise unfavorable reaction (synthetase; “energase”)

47. Purine Biosynthetic Pathway • Coupling of some reactions on pathway organizes and controls processing of substrates to products in each step • Increases overall rate of pathway and protects intermediates from degradation • In animals, IMP synthesis pathway is coupled: • Reactions 3, 4, 6 • Reactions 7, 8 • Reactions 10, 11

48. IMP Conversion to AMP

50. IMP Conversion to GMP