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FCH 532 Lecture 29

FCH 532 Lecture 29. Chapter 28: Nucleotide metabolism Chapter 24: Photosynthesis New study guide posted. Figure 26-1cd Forms of pyridoxal-5¢-phosphate. ( c ) Pyridoxamine-5¢-phosphate (PMP) and ( d ) The Schiff base that forms between PLP and an enzyme  -amino group. Page 986. Page 987.

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FCH 532 Lecture 29

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  1. FCH 532 Lecture 29 Chapter 28: Nucleotide metabolism Chapter 24: Photosynthesis New study guide posted

  2. Figure 26-1cd Forms of pyridoxal-5¢-phosphate.(c) Pyridoxamine-5¢-phosphate (PMP) and (d) The Schiff base that forms between PLP and an enzyme -amino group. Page 986

  3. Page 987

  4. Figure 26-13 The serine dehydratase reaction. Page 997 1. Formation of Ser-PLP Schiff base, 2. Removal of the -H atom of serine, 3.  elimination of OH-, 4. Hydrolysis of Schiff base, 5. Nonenzymatic tautomerization to the imine, 6. Nonenzymatic hydrolysis to form pyruvate and ammonia.

  5. B: O H H H3C-HC--C-COO- N H H C O- 2-O3PO + N CH3 H Serine hydroxymethyltransferase catalyzes PLP-dependent C-C cleavage • Catalyzes the conversion of Thr to Gly and acetaldehyde • Cleaves C-C bond by delocalizing electrons of the resulting carbanion into the conjugated PLP ring:

  6. Figure 26-54 The syntheses of alanine, aspartate, glutamate, asparagine, and glutamine. Page 1033

  7. Figure 26-58 The conversion of glycolytic intermediate 3-phosphoglycerate to serine. Conversion of 3-phosphoglycerate’s 2-OH group to a ketone Transamination of 3-phosphohydroxypyruvate to 3-phosphoserine Hydrolysis of phosphoserine to make Ser. Page 1037

  8. Purine synthesis • Purine components are derived from various sources. • First step to making purines is the synthesis of inosine monophosphate.

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

  10. Initial derivative is Inosine monophosphate (IMP) • AMP and GMP are synthesized from IMP H Hypoxanthine base O -O P O- Inosine monophosphate

  11. Inosine monophosphate (IMP) synthesis • Pathway has 11 reactions. • Enzyme 1: ribose phosphate pyrophosphokinase • Activates ribose-5-phosphate (R5P; product of pentose phosphate pathway) to 5-phosphoriobysl--pyrophosphate (PRPP) • PRPP is a precursor for Trp, His, and pyrimidines • Ribose phosphate pyrophosphokinase regualtion: activated by PPi and 2,3-bisphosphoglycerate, inhibited by ADP and GDP.

  12. Page 1071

  13. Activation of ribose-5-phosphate to PRPP N9 of purine added Page 1071

  14. Anthranilate synthase Anthranilate phosphoribosyltransferase N-(5’-phosphoribosyl) anthranilate isomerase Indole-3-glycerol phosphate synthase Tryptophan synthase Tryptohan synthase,  subunit Chorsmate mutase Prephenate dehydrogenase Aminotransferase Prephenate dehydratase aminotransferase Page 1043

  15. ATP phosphoribosyltransferase Pyrophosphohydrolase Phosphoribosyl-AMP cyclohydrolase Phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide isomerase Imidazole glycerol phosphate synthase Imidazole glycerol phosphate dehydratase L-histidinol phosphate aminotransferase Histidinol phosphate phosphatase Histidinol dehydrogenase Page 1045

  16. Page 1074

  17. Nucleoside diphosphates are synthesized by phosphorylation of nucleoside monophosphates Nucleoside diphosphates • Reactions catalyzed by nucleoside monophosphate kinases Adenylate kinase 2ADP AMP + ATP Guanine specific kinase GDP + ADP GMP + ATP • Nucleoside monophosphate kinases do not discriminate between ribose and deoxyribose in the substrate (dATP or ATP, for example)

  18. Nucleoside triphosphates are synthesized by phosphorylation of nucleoside monophosphates Nucleoside diphosphates • Reactions catalyzed by nucleoside diphosphate kinases Adenylate kinase ADP + GTP ATP + GDP • Can use any NTP or dNTP or NDP or dNDP

  19. Regulation of purine biosynthesis • Pathways synthesizing IMP, ATP and GTP are individually regulated in most cells. • Control total purines and also relative amounts of ATP and GTP. • IMP pathway regulated at 1st 2 reactions (PRPP and 5-phosphoribosylamine) • Ribose phosphate pyrophosphokinse- is inhibited by ADP and GDP • Amidophosphoribosyltransferase (1st committed step in the formation of IMP; reaction 2) is subject to feedback inhibition (ATP, ADP, AMP at one site and GTP, GDP, GMP at the other). • Amidophosphoribosyltransferase is allosterically activated by PRPP.

  20. Activation of ribose-5-phosphate to PRPP N9 of purine added Page 1071

  21. Figure 28-5 Control network for the purine biosynthesis pathway. Feedback inhibition is indicated by red arrows Feedforward activation by green arrows. Page 1075

  22. Salvage of purines • Free purines (adenine, guanine, and hypoxanthine) can be reconverted to their corresponding nucleotides through salvage pathways. • In mammals purines are salvaged by 2 enzymes • Adeninephosphoribosyltransferase (APRT) Adenine + PRPP  AMP + PPi • Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Hypoxanthine + PRPP  IMP + PPi Guanine + PRPP  GMP + PPi

  23. Synthesis of pyrimidines • Pyrimidines are simpler to synthesize than purines. • N1, C4, C5, C6 are from Asp. • C2 from bicarbonate • N3 from Gln • Synthesis of uracil monoposphate (UMP) is the first step for producing pyrimidines.

  24. Figure 28-6 The biosynthetic origins of pyrimidine ring atoms. Page 1077

  25. Page 1077

  26. Reaction 4: Oxidation of dihydroorateReactions catalyzed by eukaryotic dihydroorotate dehydrogenase. Page 1078

  27. Oxidation of dihydroorotate • Irreversible oxidation of dihydroorotate to orotate by dihydroroorotate dehydrogenase (DHODH) in eukaryotes. • In eukaryotes-FMN co-factor, located on inner mitochondrial membrane. Other enzymes for pyrimidine synthesis in cytosol. • Bacterial dihydroorotate dehydrogenases use NAD linked flavoproteins (FMN, FAD, [2Fe-2S] clusters) and perform the reverse reaction (orotate to dihydroorotate)

  28. Figure 28-9Reaction 6: Proposed catalytic mechanism for OMP decarboxylase. Decarboxylation to form UMP involves OMP decarboxylase (ODCase) to form UMP. Enhances kcat/KM of decarboxylation by 2 X 1023 No cofactors Page 1079

  29. Synthesis of UTP and CTP • Synthesis of pyrimidine nucleotide triphosphates is similar to purine nucleotide triphosphates. • 2 sequential enzymatic reactions catalyzed by nucleoside monophosphate kinase and nucleoside diphosphate kinase respectively: UMP + ATP  UDP + ADP UDP + ATP  UTP + ADP

  30. Figure 28-10 Synthesis of CTP from UTP. Page 1080 CTP is formed by amination of UTP by CTP synthetase In animals, amino group from Gln In bacteria, amino group from ammonia

  31. Regulation of pyrimidine nucleotide synthesis • Bacteria regulated at Reaction 2 (ATCase) • Allosteric activation by ATP • Inhibition by CTP (in E. coli) or UTP (in other bacteria). • In animals pyrimidine biosynthesis is controled by carbamoyl phosphate synthetase II • Inhibited by UDP and UTP • Activated by ATP and PRPP • Mammals have a second control at OMP decarboxylase (competitively inhibited by UMP and CMP) • PRPP also affects rate of OMP production, so, ADP and GDP will inhibit PRPP production.

  32. Page 1080

  33. Production of deoxyribose derivatives • Derived from corresponding ribonucleotides by reduction of the C2’ position. • Catalyzed by ribonucleotide reductases (RNRs) dADP ADP

  34. Overview of dNTP biosynthesis One enzyme, ribonucleotide reductase, reduces all four ribonucleotides to their deoxyribose derivatives. 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).

  35. Figure 28-12a Class I ribonucleotide reductase from E. coli. (a) A schematic diagram of its quaternary structure. Page 1082

  36. Proposed mechanism for rNDP reductase

  37. Proposed reaction mechanism for ribonucleotide reductase Free radical abstracts H from C3’ Acid-catalyzed cleavage of the C2’-OH bond Radical mediates stabilizationof the C2’ cation (unshared electron pair) Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical 3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

  38. Thioredoxin and glutaredoxin • Final step in the RNR catalytic cycle is the reduction of disulfide bond to reform the redox-active sulfyhydryl pair). • Thioredoxin-108 residue protein that has redox active Cys (Cys32 and Cys35)-also involved in the Calvin Cycle. • Reduces oxidized RNR and is regenerated via NADPH by thioredoxin reductase. • Glutaredoxin is an 85 residue protein that can also reduce RNR. • Oxidized glutaredoxin is reuced by NADPH using glutredeoxin reductase.

  39. Sources of reducing power for rNDP reductase

  40. Proposed reaction mechanism for ribonucleotide reductase Free radical abstracts H from C3’ Acid-catalyzed cleavage of the C2’-OH bond Radical mediates stabilizationof the C2’ cation (unshared electron pair) Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical 3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

  41. dNTPs made by phosphorylation of dNDP • Reaction is catalyzed by nucleoside diphosphate kinase (same enzyme that phosphorylates NDPs) dNDP + ATP  dNTP + ADP • Can use any NTP or dNTP as phosphoryl donor.

  42. Thymine synthesis • 2 main enzymes: dUTP diphosphohydrolase (dUTPase) and thymidylate synthase Reaction 1 • dTMP is made by methylation of dUMP. • dUMP is made by hydrolysis of dUTP via dUTP diphosphohydrolase (dUTPase) dUTP + H2O  dUMP+ PPi • Done to minimize the concentration of dUTP-prevents incorporation of uracil into DNA.

  43. Thymine synthesis Reaction 2 • dTMP is made from dUMP by thymidylate synthase (TS). • Uses N5, N10-methylene-THF as methyl donor + dUMP + dTMP

  44. Figure 28-19 Catalytic mechanism of thymidylate synthase. Enzyme Cys thiolate group attacks C6 of dUMP (nucleophile). C5 of the enolate ion attacks the CH2 group of the imium cation of N5, N10-methylene-THF. Enzyme base abstracts the acidic proton at C5, forms methylene group and eliminates THF cofactor Migration of the N6-H atom of THF to the exocyclic methylene group to form a methyl group and displace the Cys thiolate intermediate. Page 1090

  45. F FdUMP 5-flurodeoxyuridylate (FdUMP) • Antitumor agent. • Irreversible inhibitor of TS • Binds like dUMP but in step 3 of the reaction, F cannot be extracted. • Suicide substrate.

  46. Figure 28-20 The X-ray structure of the E. coli thymidylate synthase–FdUMP–THF ternary complex. Page 1091

  47. Thymine synthase oxidizes N5,N10-methyleneTHF • Only enzyme to change the oxidation state of THF. • Regenerated by 2 reactions • DHF is reduced to THF by NADPH by dihydrofolate reductase. • Serine hydroxymethyltransferase transfers the hydroxymethyl group of serine to THF to regenerate N5,N10-methylene-THF and produces glycine.

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