1FCH 532 Lecture 29 Chapter 28: Nucleotide metabolism Quiz on Mon (4/16): IMP synthesis-Purine synthesisQuiz on Wed(4/18): Pyrimidine biosynthesis/regulationQuiz on Friday(4/20): Ribonucleotide reductase mechanismFriday (4/20): extra credit seminar, Dr. Jimmy Hougland,145 Baker, 3-4PM.ACS exam has been moved to Monday (4/30)Quiz on Final is scheduled for May 4, 12:45PM-2:45PM, in 111 Marshall
6Synthesis 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 + ADPUDP + ATP UTP + ADP
7Figure 28-10 Synthesis of CTP from UTP. Page 1080CTP is formed by amination of UTP by CTP synthetaseIn animals, amino group from GlnIn bacteria, amino group from ammonia
8Regulation of pyrimidine nucleotide synthesis Bacteria regulated at Reaction 2 (ATCase)Allosteric activation by ATPInhibition by CTP (in E. coli) or UTP (in other bacteria).In animals pyrimidine biosynthesis is controled by carbamoyl phosphate synthetase IIInhibited by UDP and UTPActivated by ATP and PRPPMammals 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.
10Production of deoxyribose derivatives Derived from corresponding ribonucleotides by reduction of the C2’ position.Catalyzed by ribonucleotide reductases (RNRs)dADPADP
11Overview of dNTP biosynthesis One enzyme, ribonucleotide reductase,reduces all four ribonucleotides to theirdeoxyribose derivatives.A free radical mechanism is involvedin the ribonucleotide reductasereaction.There are three classes of ribonucleotidereductase enzymes in nature:Class I: tyrosine radical, uses NDPClass II: adenosylcobalamin. uses NTPs(cyanobacteria, some bacteria,Euglena).Class III: SAM and Fe-S to generateradical, uses NTPs.(anaerobes and fac. anaerobes).
12Figure 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
14Proposed reaction mechanism for ribonucleotide reductase Free radical abstracts H from C3’Acid-catalyzed cleavage of the C2’-OH bondRadical mediates stabilizationof the C2’ cation (unshared electron pair)Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.
15Thioredoxin 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.
17Proposed reaction mechanism for ribonucleotide reductase Free radical abstracts H from C3’Acid-catalyzed cleavage of the C2’-OH bondRadical mediates stabilizationof the C2’ cation (unshared electron pair)Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.
18dNTPs made by phosphorylation of dNDP Reaction is catalyzed by nucleoside diphosphate kinase (same enzyme that phosphorylates NDPs)dNDP + ATP dNTP + ADPCan use any NTP or dNTP as phosphoryl donor.
19Thymine synthesis2 main enzymes: dUTP diphosphohydrolase (dUTPase) and thymidylate synthaseReaction 1dTMP is made by methylation of dUMP.dUMP is made by hydrolysis of dUTP via dUTP diphosphohydrolase (dUTPase)dUTP + H2O dUMP+ PPiDone to minimize the concentration of dUTP-prevents incorporation of uracil into DNA.
20Thymine synthesis + dUMP + dTMP Reaction 2 dTMP is made from dUMP by thymidylate synthase (TS).Uses N5, N10-methylene-THF as methyl donor+dUMP+dTMP
21Figure 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 cofactorMigration 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
225-flurodeoxyuridylate (FdUMP) Antitumor agent.Irreversible inhibitor of TSBinds like dUMP but in step 3 of the reaction, F cannot be extracted.Suicide substrate.FdUMPF
23Figure 28-20. The X-ray structure of the E Figure The X-ray structure of the E. coli thymidylate synthase–FdUMP–THF ternary complex.Page 1091
24Thymine synthase oxidizes N5,N10-methyleneTHF Only enzyme to change the oxidation state of THF.Regenerated by 2 reactionsDHF 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.
25Figure 28-21 Regeneration of N5,N10-methylenetetrahydrofolate. Page 1091
26Nucleotide degradation Nucleic acids can survive the acid of the stomachDegraded into nucleotides by pancreatic nucleases and intestinal phosphodiesterases in the duodenum.Components cannot pass through cell membranes, so they are hydrolyzed to nucleosides.Nucleosides may be directly absorbed by the intestine or undergo further degradation to free bases and ribose or ribose-1-phosphate by nucleosidases and nucloside phosphorylase.Nucleoside + H2O base + riboseNucleoside + Pi base + ribose-1-PnucleosidaseNucleoside phosphorylase
27Purine nucleoside + Pi Purine base + ribose-1-P Catabolism of purinesAll pathways lead to formation of uric acid.Intermediates could be intercepted into salvage pathways.1st reaction is the nucleotidase and second is catalyzed by purine nucleoside phosphorylase (PNP)Ribose-1-phosphate is isomerized by phosphoribomutase to ribose-5-phosphate (precursor to PRPP).Purine nucleoside + Pi Purine base + ribose-1-PAdenosine and deoxyadenosine are not degraded by PNP but are deaminated by adenosine deaminase (ADA) and AMP deaminase in mammalsPurine nucleoside phosphorylase
28Figure 28-23 Major pathways of purine catabolism in animals. ADAGenetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese (SCID).No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPsPage 1093
29Figure 28-24a. Structure and mechanism of adenosine deaminase Figure 28-24a Structure and mechanism of adenosine deaminase. (a) A ribbon diagram of murine adenosine deaminase in complex with its transition state analog HDPR.Page 1094
30Figure 28-24b (b) The proposed catalytic mechanism of adenosine deaminase. Zn2+ polarized H2O molecule nucleophilically attacks C6 of the adenosine. His is general base catalyst, Glu is general acid, and Asp orients water.Results in tetrahedral intermediate which decomposes by elimination of ammonia.Product is inosine in enol form (assumes dominant keto form upon release from enzyme).Page 1094
31Purine nucleotide cycle Deamination of AMP to IMP combined with synthesis of AMP from IMP results in deaminating Asp to yield fumarate.Important role in skeletal muscle-increased activity requires increased activity in the citric acid cycle.Muscle replenishes citric acid cycle intermediates through the purine nucleotide cycle.
32Figure 28-25 The purine nucleotide cycle. Page 1095
33Xanthine oxidaseXanthine oxidse (XO) converts hypoxanthine to xanthine, and xanthine to uric acid.In mammals, found in the liver and small intestine mucosaXO is a homodimer with FAD, two [2Fe-2S] clusters and a molybdopterin complex (Mo-pt) that cycles between Mol (VI) and Mol (IV) oxidation states.Final electron acceptor is O2 which is converted to H2O2XO is cleaved into 3 segments. The uncleaved enzyme is known as xanthine dehydrogenase (uses NAD+ as an electron acceptor where XO does not).XO hydroxylates hypoxanthine at its C2 position and xanthine at the C8 positon to produce uric acid in the enol form.
34N-terminal domain is cyan Figure 28-26a X-Ray structure of xanthine oxidase from cow’s milk in complex with salicylic acid.N-terminal domain is cyanCentral domain is goldC-terminal domain is lavenderPage 1095
35Mechanism for XOReaction initiated by attack of enzyme nucleophile on the C8 position of xanthine.The C8-H atom is eliminated as a hydride ion that combines with Mo (VI) complex, reducing it to Mo (IV).Water displaces the enzyme nucleophile producing uric acid.
36Figure 28-27 Mechanism of xanthine oxidase. Page 1096
37Figure 28-23 Major pathways of purine catabolism in animals. ADAGenetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese (SCID).No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPsPage 1093
38Goal is the conservation of water. Purine degredation in other animalsPrimates, birds, reptiles, insects-final degradation product id uric acid which is excreted in urine.Goal is the conservation of water.
39Figure 28-29 The Gout, a cartoon by James Gilroy (1799). Page 1097Gout is a disease characterized by elevated levels of uric acid in body fluids. Caused by deposition of nearly insoluble crystals of sodium urate or uric acid.
40Clinical disorders of purine metabolism Excessive accumulation of uric acid: GoutThe three defects shown each result in elevated de novo purine biosynthesis
41Common treatment for gout: allopurinol Allopurinol is an analogue of hypoxanthine that strongly inhibitsxanthine oxidase. Xanthine and hypoxanthine, which are soluble, are accumulated and excreted.
42Catabolism of pyrimidines Animal cells degrade pyrimidines to their component bases.Happen through dephosphorylation, deamination, and glycosidic bond cleavage.Uracil and thymine broken down by reduction (vs. oxidation in purine catabolism).
44Biosynthesis of of NAD and NADP+ Produced from vitamin precursors Nicotinate and Nicotinamide and from quinolinate, a Trp degradation productPage 1099
45Biosynthesis of FMN and FAD from riboflavin FAD is synthesized from riboflavin in a two-reaction pathway.Flavokinase phosphorylates the 5’OH group to give FMNFAD pyrophosphorylase catalyzes the next step (coupling of FMN to ADP).Page 1100