Transcript Lecture 27
FCH 532 Lecture 29 Chapter 28: Nucleotide metabolism Chapter 24: Photosynthesis New study guide posted 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 Figure 26-1cd Page 987 Page 997 Figure 26-13 The serine dehydratase reaction. 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. 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: H B: O H H3C-HC--C-COON H C 2-O H O- 3PO + N H CH3 Page 1033 Figure 26-54 The syntheses of alanine, aspartate, glutamate, asparagine, and glutamine. Page 1037 Figure 26-58 The conversion of glycolytic intermediate 3phosphoglycerate to serine. 1. Conversion of 3phosphoglycerate’s 2-OH group to a ketone 2. Transamination of 3phosphohydroxypyruvate to 3-phosphoserine 3. Hydrolysis of phosphoserine to make Ser. Purine synthesis • • Purine components are derived from various sources. First step to making purines is the synthesis of inosine monophosphate. De novo biosynthesis of purines: low molecular weight precursors of the purine ring atoms Initial derivative is Inosine monophosphate (IMP) • AMP and GMP are synthesized from IMP H O -O P O- Inosine monophosphate Hypoxanthine base 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. Page 1071 Page 1071 1. Activation of ribose-5phosphate to PRPP 2. N9 of purine added Anthranilate synthase 2. Anthranilate phosphoribosyltrans ferase 3. N-(5’phosphoribosyl) anthranilate isomerase 4. Indole-3-glycerol phosphate synthase 5. Tryptophan synthase 6. Tryptohan synthase, subunit Page 1043 1. 7. Chorsmate mutase 8. Prephenate dehydrogenase 9. Aminotransferase 10. Prephenate dehydratase 11. aminotransferase Page 1045 1. ATP phosphoribosyltransferase 2. Pyrophosphohydrolase 3. Phosphoribosyl-AMP cyclohydrolase 4. Phosphoribosylformimino-5aminoimidazole carboxamide ribonucleotide isomerase 5. Imidazole glycerol phosphate synthase 6. Imidazole glycerol phosphate dehydratase 7. L-histidinol phosphate aminotransferase 8. Histidinol phosphate phosphatase 9. Histidinol dehydrogenase Page 1074 Nucleoside diphosphates are synthesized by phosphorylation of nucleoside monophosphates Nucleoside diphosphates • Reactions catalyzed by nucleoside monophosphate kinases Adenylate kinase AMP + ATP 2ADP Guanine specific kinase GMP + ATP • GDP + ADP Nucleoside monophosphate kinases do not discriminate between ribose and deoxyribose in the substrate (dATP or ATP, for example) Nucleoside triphosphates are synthesized by phosphorylation of nucleoside monophosphates Nucleoside diphosphates • Reactions catalyzed by nucleoside diphosphate kinases Adenylate kinase ATP + GDP • ADP + GTP Can use any NTP or dNTP or NDP or dNDP 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. Page 1071 1. Activation of ribose-5phosphate to PRPP 2. N9 of purine added Figure 28-5 Control network for the purine biosynthesis pathway. Page 1075 Feedback inhibition is indicated by red arrows Feedforward activation by green arrows. 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 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. Page 1077 Figure 28-6 The biosynthetic origins of pyrimidine ring atoms. Page 1077 Page 1078 Reaction 4: Oxidation of dihydroorate Reactions catalyzed by eukaryotic dihydroorotate dehydrogenase. 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) Page 1079 Figure 28-9 Reaction 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 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 Page 1080 Figure 28-10 Synthesis of CTP from UTP. CTP is formed by amination of UTP by CTP synthetase In animals, amino group from Gln In bacteria, amino group from ammonia 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. • • • • Page 1080 Production of deoxyribose derivatives • • Derived from corresponding ribonucleotides by reduction of the C2’ position. Catalyzed by ribonucleotide reductases (RNRs) ADP dADP 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). Page 1082 Figure 28-12a Class I ribonucleotide reductase from E. coli. (a) A schematic diagram of its quaternary structure. Proposed mechanism for rNDP reductase Proposed reaction mechanism for ribonucleotide reductase 1. Free radical abstracts H from C3’ 2. Acid-catalyzed cleavage of the C2’-OH bond 3. Radical mediates stabilizationof the C2’ cation (unshared electron pair) 4. Radical-cation intermediate is reduced by redoxactive sulhydryl pairdeoxynucleotide radical 5. 3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state. 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. Sources of reducing power for rNDP reductase Proposed reaction mechanism for ribonucleotide reductase 1. Free radical abstracts H from C3’ 2. Acid-catalyzed cleavage of the C2’-OH bond 3. Radical mediates stabilizationof the C2’ cation (unshared electron pair) 4. Radical-cation intermediate is reduced by redoxactive sulhydryl pairdeoxynucleotide radical 5. 3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state. 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. 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. Thymine synthesis Reaction 2 • dTMP is made from dUMP by thymidylate synthase (TS). • Uses N5, N10-methylene-THF as methyl donor + dUMP + dTMP Page 1090 Figure 28-19 Catalytic mechanism of thymidylate synthase. 1. Enzyme Cys thiolate group attacks C6 of dUMP (nucleophile). 2. C5 of the enolate ion attacks the CH2 group of the imium cation of N5, N10-methyleneTHF. 3. Enzyme base abstracts the acidic proton at C5, forms methylene group and eliminates THF cofactor 4. Migration of the N6-H atom of THF to the exocyclic methylene group to form a methyl group and displace the Cys thiolate intermediate. 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. F FdUMP Page 1091 Figure 28-20 The X-ray structure of the E. coli thymidylate synthase–FdUMP–THF ternary complex. Thymine synthase oxidizes N5,N10methyleneTHF • • • • 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,N10methylene-THF and produces glycine.