FCH 532 Lecture 28 Chapter 28: Nucleotide metabolism Quiz on Monday essential amino acids Wed. April 11-Exam 3 ACS exam is on Monday 5/30 Final is scheduled for May 4, 12:45-2:45 PM, in 111 Marshall

Figure 26-60 The biosynthesis of the “aspartate family” of amino acids: lysine, methionine, and threonine. Page 1039

Figure 26-61 The biosynthesis of the “pyruvate family” of amino acids: isoleucine, leucine, and valine. Page 1040

Figure 26-62 The biosynthesis of chorismate, the aromatic amino acid precursor. Page 1042

Figure 26-63 The biosynthesis of phenylalanine, tryptophan, and tyrosine from chorismate. Page 1043

Figure 26-64 A ribbon diagram of the bifunctional enzyme tryptophan synthase from S. typhimurium Page 1044

Figure 26-65 The biosynthesis of histidine. Page 1045

Hypoxanthine

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 Hypoxanthine base -O P O- Inosine monophosphate

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.

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

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

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

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Nucleoside diphosphates are synthesized by phosphorylation of nucleoside monophosphates 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)

Can use any NTP or dNTP or NDP or dNDP 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.

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

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

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.

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

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Reaction 4: Oxidation of dihydroorate Reactions catalyzed by eukaryotic dihydroorotate dehydrogenase. Page 1078

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)

Enhances kcat/KM of decarboxylation by 2 X 1023 No cofactors 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 Page 1079

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

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

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.

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

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).

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

Proposed mechanism for rNDP reductase

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.