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

Figure 26-1cdForms 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

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Figure 26-13The serine dehydratase reaction. Page 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: +N+N H CH 3 2- O 3 PO C N H H O-O- H 3 C-HC  --C  -COO - O H H B:

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

Figure 26-58The conversion of glycolytic intermediate 3- phosphoglycerate to serine. Page Conversion of 3- phosphoglycerate’s 2-OH group to a ketone 2.Transamination of 3- phosphohydroxypyruvate 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 P O- -O O Hypoxanthine base 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 PP i and 2,3-bisphosphoglycerate, inhibited by ADP and GDP.

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1.Activation of ribose-5- phosphate to PRPP 2.N9 of purine added

Page 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 7.Chorsmate mutase 8.Prephenate dehydrogenase 9.Aminotransferase 10.Prephenate dehydratase 11.aminotransferase

Page ATP phosphoribosyltransferase 2.Pyrophosphohydrolase 3.Phosphoribosyl-AMP cyclohydrolase 4.Phosphoribosylformimino-5- aminoimidazole 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

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Nucleoside diphosphates are synthesized by phosphorylation of nucleoside monophosphates Nucleoside diphosphates Reactions catalyzed by nucleoside monophosphate kinases AMP + ATP 2ADP Adenylate kinase GMP + ATP GDP + ADP Guanine specific kinase 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 ATP + GDP ADP + GTP Adenylate kinase 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 Activation of ribose-5- phosphate 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 + PP i Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Hypoxanthine + PRPP  IMP + PP i Guanine + PRPP  GMP + PP i

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

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)

Figure 28-9Reaction 6: Proposed catalytic mechanism for OMP decarboxylase. Page 1079 Decarboxylation to form UMP involves OMP decarboxylase (ODCase) to form UMP. Enhances k cat /K M of decarboxylation by 2 X 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

Figure 28-10Synthesis 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) 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).

Figure 28-12aClass 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 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 redox- active sulhydryl pair- deoxynucleotide 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 redox- active sulhydryl pair- deoxynucleotide 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 + H 2 O  dUMP+ PP i 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 Enzyme Cys thiolate group attacks C6 of dUMP (nucleophile). 2.C5 of the enolate ion attacks the CH 2 group of the imium cation of N 5, N 10 -methylene- THF. 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. Figure 28-19Catalytic mechanism of thymidylate synthase.

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. FdUMP F

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

Thymine synthase oxidizes N 5,N 10 - 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 N 5,N 10 - methylene-THF and produces glycine.