Chapter 26

1. Chapter 26 The Synthesis and Degradation of Nucleotides Biochemistry by Reginald Garrett and Charles Grisham

2. Outline • Can Cells Synthesize Nucleotides? • How Do Cells Synthesize Purines? • Can Cells Salvage Purines? • How Are Purines Degraded? • How Do Cells Synthesize Pyrimidines? • How Are Pyrimidines Degraded? • How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? • How Are Thymine Nucleotides Synthesized?

3. Nucleotides and Nucleic Acids • Nucleotides: (nucleoside + phosphate) • Biological molecules that possess a heterocyclic nitrogenous base, a five-carbon sugar (ribose), and phosphate as principal components (chapter 10) • Participate as essential intermediates in cellular metabolisms—NAD, FAD, ATP, cAMP… • The elements of heredity and the agents of genetic information transfer—nucleic acids • Nucleic acids: • Nucleotides are the monomeric units of nucleic acid • Two basic kinds of nucleic acids • Deoxyribonucleic acid (DNA) • Ribonucleic acid (RNA)

4. 26.1 – Can Cells Synthesize Nucleotides? • Nearly all organisms synthesize purines and pyrimidines "de novo biosynthesis pathway“ • Many organisms also "salvage" purines and pyrimidines from diet and degradative pathways • Ribose can be catabolized to generate energy, but nitrogenous bases do not • Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies

5. 26.2 – How Do Cells Synthesize Purines? John Buchanan (1948) "traced" the sources of all nine atoms of purine ring • N-1: aspartic acid • N-3, N-9: glutamine • C-2, C-8: N10-formyl-THF - one carbon units • C-4, C-5, N-7: glycine • C-6: CO2

6. Figure 26.3 The de novo pathway for purine synthesis. Step 1: Ribose-5-phosphate pyrophosphokinase. Step 2: Glutamine phosphoribosyl pyrophosphate amidotransferase. Step 3: Glycinamide ribonucleotide (GAR) synthetase. Step 4: GAR transformylase. Step 5: FGAM synthetase (FGAR amidotransferase). Step 6: FGAM cyclase (AIR synthetase). Step 7: AIR carboxylase. Step 8: SAICAR synthetase. Step 9: adenylosuccinase. Step 10: AICAR transformylase. Step 11: IMP synthase.

7. IMP Biosynthesis IMP (inosinic acid or inosine monophosphate) is the immediate precursor to GMP and AMP First step: Ribose-5-phosphate pyrophosphokinase • PRPP synthesis from ribose-5-phosphate and ATP • PRPP is limiting substance for purine synthesis • But PRPP is a branch point so next step is the committed step (fig 26.6) Second step: Gln PRPP amidotransferase • Form phosphoribosyl-b-amine; Changes C-1 configuration (a→b) • GMP and AMP inhibit this step - but at distinct sites • Azaserine - Glutamine analog - inhibitor/anti-tumor

8. Figure 26.4 The structure of azaserine. Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site.

9. Step 3: Glycinamide ribonucleotide (GAR) synthetase • Glycine carboxyl condenses with amine in two steps • Glycine carboxyl activated by -P from ATP • Amine attacks glycine carboxyl • Synthesize glycinamide ribonucleotide Step 4: Glycinamide ribonucleotide (GAR) transformylase • Formyl group of N10-formyl-THF is transferred to free amino group of GAR • Yield N-Formylglycinamide ribonucleotide

12. Step 5: Formylglycinamide ribonucleotide (FGAR) amidotransferase (FGAM synthetase) • Formylglycinamidine ribonucleotide(FGAM) • C-4 carbonyl forms a P-ester from ATP and active NH3 attacks C-4 to form imine • Irreversibly inactivated by azaserine

13. Closure of the first ring, carboxylation and attack by aspartate Step 6: FGAM cyclase (AIR synthetase) • Produce aminoimidazole nucleotide (AIR) • Similar in some ways to step 5. ATP activates the formyl group by phosphorylation, facilitating attack by N. • In avian liver, the enzymes for step 3, 4, and 6 (GAR synthetase, GAR transformylase, and AIR synthetase) reside on a polypeptide

14. Step 7: AIR carboxylase • The product is carboxyaminoimidazole ribonucleotide (CAIR) • Carbon dioxide is added in ATP-dependent reaction Step 8: SAICAR synthetase • N-succinylo-5-aminoimidazole-4-carboxamide ribonucleotide • Attack by the amino group of aspartate links this amino acid with the carboxyl group • The enzymes for steps 7 and 8 reside on a bifunctional polypeptide in avian

16. Step 9: Adenylosuccinase (also see Fig 26.5) • The product is 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR); remove fumarate • AICAR is also an intermediate in the histidine biosynthetic pathway Step 10: AICAR transformylase • N-formylaminoimidazole-4-carboxamide ribonucleotide (FAICAR) • Another 1-C addition (N10-formyl-THF) Step 11: IMP synthase(IMP cyclohydrolase) • Amino group attacks formyl group to close the second ring • The enzymes for steps 10 and 11 reside on a bifunctional polypeptide in avian

18. 6 ATPs are required in the purine biosynthesis from ribose-5-phosphate to IMP, but that this is really 7 ATP equivalents • The dependence of purine biosynthesis on THF (tetrahydrofolate) in two steps means that methotrexate and sulfonamides block purine synthesis

19. Tetrahydrofolate and One-Carbon Units • Folic acid, a B vitamin found in green plants, fresh fruits, yeast, and liver, is named from folium, Latin for “leaf”. • Folates are acceptors and donors of one-carbon units for all oxidation levels of carbon except CO2 (for which biotin is the relevant carrier). • The active form is tetrahydrofolate.

20. Tetrahydrofolate and One-Carbon Units Folates are acceptors and donors of one-carbon units for all oxidation levels of carbon except CO2 (for which biotin is the relevant carrier).

21. Tetrahydrofolate and One-Carbon Units Oxidation numbers are calculated by assigning valence bond electrons to the more electronegative atom and then counting the charge on the quasi ion. A carbon assigned four valence electrons would have an oxidation number of 0. The carbon in N5-methyl-THF (top left) is assigned six electrons from the three C-H bonds and thus has a oxidation number of -2.

23. Folate Analogs as Antimicrobial and Anticancer Agents • De novo purine biosynthesis depends on folic acid compounds at steps 4 and 10 • For this reason, antagonists of folic acid metabolism indirectly inhibit purine formation and, in turn, nucleic acid synthesis, cell growth, and cell development • Rapidly growing cells, such as infective bacteria and fast-growing tumors, are more susceptible to such agents • Sulfonamides are effective anti-bacterial agents • Methotrexate and aminopterin are folic acid analogs that have been used in cancer chemotherapy

24. Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to p-aminobenzoate (PABA),an important precursor in folic acid synthesis. Sulfonamides block folic acid formation by competing with PABA.

25. AMP and GMP are Synthesized from IMP Figure 26.5 The synthesis of AMP and GMP from IMP.

26. AMP and GMP are synthesized from IMP IMP is the precursor to both AMP and GMP • AMP synthesis Step 1:Adenylosuccinate synthetase • The 6-O of inosine is displaced by aspartate to yield adenylosuccinate • GTP is the energy input for AMP synthesis, whereas ATP is energy input for GMP Step 2: Adenylosuccinase (adenylosuccinate lyase) • Carries out the nonhydrolytic removal of fumarate from adenylosuccinate, leaving AMP. • The same enzyme catalyzing Step 9 in the purine pathway

27. GTP synthesis Step 1: IMP dehydrogenase • Oxidation at C-2 • NAD+-dependent oxidation • xanthosine monophosphate (XMP) Step 2: GMP synthetase • Replacement of the O by N (from Gln) • ATP-dependent reaction; PPi • Starting from ribose-5-phosphate • 8 ATP equivalents are consumed in the AMP synthesis • 9 ATP equivalents in GMP synthesis

28. The regulation of purine synthesis Reciprocal control occurs in two ways IMP synthesis: Allosterically regulated at the first two steps • R-5-P pyrophosphokinase: • ADP & GDP • Phosphoribosyl pyrophosphate amidotransferase • A “series”: AMP, ADP, and ATP • G “series”: GMP, GDP, and GTP • PRPP is “feed-forward” activator

29. AMP synthesis: • adenylosuccinate synthetase is feedback-inhibited by AMP • GMP synthesis: • IMP dehydrogenase is feedback-inhibited by GMP

30. Nucleoside diphosphate and triphosphate Nucleoside diphosphate: ATP-dependent kinase • Adenylate kinase: AMP +ATP →ADP +ADP • Guanylate kinase:GMP +ATP →GDP +ADP Nucleoside triphosphate: non-specific enzyme • Nucleoside diphosphate kinase GDP +ATP GTP +ADP NDP +ATP NTP +ADP (N=G, C, U, and T)

31. 26.3 – Can Cells Salvage Purines? Salvage pathways • Nucleic acid turnover (synthesis and degradation) is an ongoing metabolic process • mRNA in particular is actively synthesized and degraded • Lead to release of free purines; adenine, guanine, and hypoxanthine (the base in IMP; Fig 26.8) • Salvage pathways exist to recover them in useful form • Involve resynthesis of nucleotides from bases via phosphoribosyltransferases (PRT)

32. 26.3 – Can Cells Salvage Purines? Base + PRPP Nucleoside-5’-phosphate + PPi The purine phosphoribosyltransferases are adenine phosphoribosyltransferases (APRT) and hypoxanthine-guanine phosphoribosyltransferases (HGPRT) Collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction (Fig 26.7) Absence of HGPRT is cause of Lesch-Nyhan syndrome (sex-linked); In Lesch-Nyhan, purine synthesis is increased 200-fold and uric acid is elevated in blood

33. Hyperxanthine-Guanine PhosphoRibosylTransferase Figure 26.7 Purine salvage by the HGPRT reaction.

34. Victims of Lesch-Nyhan syndrome experience severe arthritis due to accumulation of uric acid, as well as retardation, and other neurological symptoms.

35. 26.4 – How Are Purines Degraded? Purine catabolism leads to uric acid • Nucleotidases and nucleosidases release ribose and phosphates and leave free bases • Nucleotidase: NMP + H2O → nucleoside + Pi • Nucleosidase: nucleoside + H2O → base + ribose • PNP: nucleoside + Pi → base + ribose-P • The PNP products are converted to xanthine by xanthine oxidase and guanine deaminase • Xanthine oxidase converts xanthine to uric acid • Note that xanthine oxidase can oxidize two different sites on the purine ring system • Neither adenosine nor deoxyadenosine is a substrate for PNP • Converted to inosine by adenosine deaminase (ADA)

36. Figure 26.8 The major pathways for purine catabolism in animals. Catabolism of the different purine nucleotides converges in the formation of uric acid.

37. Severe combined immunodeficiency syndrome (SCID) The effect of elevated levels of deoxyadenosine on purine metabolism. If ADA is deficient or absent, deoxyadenosine is not converted into deoxyinosine as normal (see Figure 26.8). Instead, it is salvaged by a nucleoside kinase, which converts it to dAMP, leading to accumulation of dATP and inhibition of deoxynucleotide synthesis (see Figure 26.24). Thus, DNA replication is stalled.

38. The purine nucleoside cycle in skeletal muscle Serve as an anaplerotic pathway • Convert aspartate to fumarate plus NH4+ Figure 26.9 The purine nucleoside cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle.

39. Xanthine Oxidase and Gout • Xanthine Oxidase in liver, intestines mucosa, and milk can oxidize hypoxanthine to xanthine and xanthine to uric acid • Humans and other primates excrete uric acid in the urine, but most N goes out as urea • Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound • Gout occurs from accumulation of uric acid crystals in the extremities • Allopurinol, which inhibits xanthine oxidase , is a treatment

40. Figure 26.11 Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase. Figure 26.10 Xanthine oxidase catalyzes a hydroxylase-type reaction.

41. Animals other than humans oxidize uric acid to form excretory products • Urate oxidase: Allantoin • Allantoinase: Allantoic acid • Allantoicase: Urea • Urease: Ammonia Figure 26.12 The catabolism of uric acid to allantoin, allantoic acid, urea, or ammonia in various animals.

42. 26.5 – How Do Cells Synthesize Pyrimidines? • In contrast to purines, pyrimidines are not synthesized as nucleotides • The pyrimidine ring is completed before a ribose-5-P is added • Carbamoyl-P and aspartate are the precursors of the six atoms of the pyrimidine ring

43. Figure 26.15 The de novo pyrimidine biosynthetic pathway.

44. de novo Pyrimidine Synthesis • Step 1:Carbamoyl Phosphate synthesis • Carbamoyl phosphate for pyrimidine synthesis is made by carbamoyl phosphate synthetase II (CPS II) • This is a cytosolic enzyme (whereas CPS I is mitochondrial and used for the urea cycle) • Substrates are HCO3-, glutamine (not NH4+),2 ATP • In mammals, CPS-II can be viewed as the committed step in pyrimidine synthesis • Bacteria have but one CPS; thus, the committed step is the next reaction, which is mediated by aspartate transcarbamoylase (ATCase)

45. (also called carbonyl-phosphate) Figure 26.14The reaction catalyzed by carbamoyl phosphate synthetase II (CPS II).

46. Step 2: Aspartate transcarbamoylase (ATCase) • catalyzes the condensation of carbamoyl phosphate with aspartate to form carbamoyl-aspartate • carbamoyl phosphate represents an ‘activated’ carbamoyl group • Step 3: dihydroorotase • ring closure and dehydration via intramolecular condensation • Produce dihydroorotate

47. Step 4: dihydroorotate dehydrogenase • Synthesis of a true pyrimidine (orotate) • Step 5: orotate phosphoribosyltransferase • Orotate is joined with a ribose-P to form orotidine-5’-phosphate (OMP) • The ribose-P donor is PRPP • Step 6: OMP decarboxylase • OMP decarboxylase makes UMP (uridine-5’-monophposphate, uridylic acid)

48. Metabolic channeling • In bacteria, the six enzymes are distinct • Eukaryotic pyrimidine synthesis involves channeling and multifunctional polypeptides • CPS-II, ATCase, and dihydroorotase are on a cytosolic polypeptide • Orotate PRT and OMP decarboxylase on the other cytosolic polypeptide (UMP synthase) • The metabolic channeling is more efficient

49. UTP and CTP • Nucleoside monophosphate kinase UMP + ATP → UDP + ADP • Nucleoside diphosphate kinase UDP + ATP → UTP + ADP • CTP sythetase forms CTP from UTP and ATP

50. Regulation of pyrimidine biosynthesis • In bacteria • allosterically inhibited at ATCase by CTP (or UTP) • allosterically activated at ATCase by ATP (compete with CTP) • In animals • UDP and UTP are feedback inhibitors of CPS II • PRPP and ATP are allosteric activators