Derivatives of Amino Acids and Metabolism of Nucleotides CH353 January 29, 2008

Anabolic Role of the Citric Acid Cycle Error: glycine not glutamate provides carbons for purines Purines X Biosynthesis of amino acids & derivatives from citric acid cycle intermediates require anaplerotic reactions (red arrows) for replenishing metabolites

Derivatives of Amino Acids Porphyrins and Heme Glycine + Succinyl-CoA (animals) Glutamate (bacteria & plants) Non-ribosomal peptide synthesis peptidoglycan, antibiotics glutathione (glutamate + cysteine + glycine) Modified amino acids plant compounds, neurotransmitters, polyamines Nucleotide heterocyclic bases purines and pyrimidines

Biosynthesis of Heme animals heme precursor bacteria, plants

Biosynthesis of Heme

Genetic Deficiencies in Heme Biosynthesis

Catabolism of Heme purple Regulated step: 3 isozymes green yellow Important serum antioxidant Bile pigment yellow (oxidized) red-brown (reduced)

Reactions with Monooxygenases Use 2 reductants for O2 (mixed-function oxygenases) One reductant accepts an O atom Other reductant provides 2 H’s to the second O atom General Reaction: AH + BH2 + O–O → A–OH + B + H2O

Biosynthesis of Nitric Oxide NO involved in intercellular signaling NO synthase (a mixed-function oxygenase) dimer, similar to NADPH cytochrome P450 reductase cofactors: FMN, FAD, tetrahydrobiopterin, Fe3+ heme catalyzes a 5 e- oxidation

Biosynthesis of Creatine metabolite for storage of high energy transfer potential phosphate phosphorylated at high [ATP] amidinotransferase exchanges amino acids glycine for ornithine 1 substrate and 1 product same as for arginase reaction except different amidino group acceptor glycine instead of water S-adenosylmethionine methyl donor

Biosynthesis of Glutathione reducing agent (redox buffer) non-ribosomal peptide synthesis carboxyl groups activated with ATP (acyl phosphate intermediates)

Non-ribosomal Peptide Synthesis Microbial peptides are synthesized by multi-modular synthases; similar to fatty acid biosynthesis Modular complexes of enzymes for recognition, activation, modification and condensation of a specific amino acid to the growing polymer Features use of unusual amino acids, D-enantiomers, and non-α peptide bonds Peptidoglycans, antibiotics and ionophores

Reactions with Pyridoxal Phosphate Amino acid racemase reactions L-alanine ↔ D-alanine Inhibitors of alanine racemase: Antibiotics – peptidoglycan biosynthesis

Biosynthesis of Plant Compounds phenylalanine, tyrosine, tryptophan precursors for plant compounds: lignin (phenolic polymer) indole-3-acetate (auxin) tannins alkaloids, e.g. morphine flavors, e.g. cinnamon, nutmeg, cloves, vanilla, cayenne pepper

Reactions with Pyridoxal Phosphate Decarboxylase reactions Histidine → Histamine + CO2 Ornithine → Putrescine + CO2

Biosynthesis of Neurotransmitters Pathways involve decarboxylases and mixed-function oxygenases (monooxygenases)

Biosynthesis of Spermidine and Spermine Pathway involves decarboxylases and S-adenosylmethione alkylation

African Sleeping Sickness Caused by Trypanosoma brucei rhodesiense Vaccines are ineffective: repeated change of coat antigen Therapy based on inhibitor of polyamine biosynthesis

Mechanism of Ornithine Decarboxylase

Inhibition of Ornithine Decarboxylase DMF-Ornithine

Study Problem Antihistamines are compounds that block histamine synthesis or binding to its receptor Histamine is synthesized from histidine by a pyridoxal phosphate dependent decarboxylase Design an antihistamine drug candidate, based upon the mechanism for decarboxylation Show the structure and its proposed mechanism of action

Overview of Nucleotide Metabolism Nucleotide functions Activated precursors for synthesis of RNA, DNA and cofactors Activation of biosynthetic precursors Energy for cellular processes Signal transduction Biosynthetic pathways de novo synthesis of purines and pyrimidines differ in order of attachment of ribose to base salvage pathways reacting a base with activated 5-phosphoribose (PRPP)

Precursors for Nucleotide Biosynthesis 5-phosphoribosyl-1-pyrophosphate ribose phosphate pyrophosphokinase Ribose 5-phosphate + ATP → 5-phosphoribosyl-1-pyrophosphate + AMP

Precursors for Nucleotide Biosynthesis Tetrahydrofolate (H4 folate) derivatives N5,N10-methylene-H4 folate thymidylate biosynthesis N5-formyl-H4 folate purine biosynthesis

Precursors for Nucleotide Biosynthesis Amino Acids Glycine for purine biosynthesis Aspartate for pyrimidine biosynthesis Amino Acid Nitrogen α-amino group of aspartate (purines) aspartate + [acceptor] + ATP → succinyl-amino-[acceptor] + ADP + Pi succinyl-amino-[acceptor] → amino-[acceptor] + fumarate amide group of glutamine (purines, pyrimidines) glutamine + [acceptor] + ATP → amino-[acceptor] + glutamate + ADP + Pi

Activation of Amino Acceptors carboxylate or carbonyl acceptor are activated with ATP acyl-phosphate or phospho-enol intermediates formed nucleophilic substitution of phosphate with amino group R–C–O– O R–C–OPO3–2 O R–C–NHR’ O ATP ADP R’NH2 PO4–2 C–C–R O H C–C–R OPO3–2 C–C–R NHR’

Biosynthesis of the Purine Ring Multi-step synthesis from many precursors (numbers indicate order of addition to purine ring from PRPP) 1 2 3 4 5 6 7

Purine Biosynthesis glutamine-PRPP amidotransferase GAR synthetase glutamine donates amide nitrogen to activated 5-phosphoribose (PRPP) committed step for purine synthesis product unstable t½ = 30 seconds GAR synthetase glycine carboxyl activated with ATP Pi displaced; amide bond formed GAR transformylase N10-formyl tetrahydrofolate donates formyl group to glycine amino group FGAR amidotransferase ATP activates carbonyl group amidotransfer displaces Pi

Purine Biosynthesis FGAM cyclase (AIR synthetase) in bacteria & fungi: ATP activates carbonyl cyclization of imidazole ring in bacteria & fungi: N5-CAIR synthetase ATP activates HCO3- carbamoylation of exocyclic amine N5-CAIR mutase transfer of carboxylate to ring in higher eukaryotes: AIR carboxylase formation of only C-C bond no cofactors or ATP required

Purine Biosynthesis SAICAR synthetase SAICAR lyase aspartate is amino donor ATP activates carboxylate aspartate amino replaces Pi SAICAR lyase fumarate is eliminated steps 8 & 9 analogous to urea cycle AICAR from histidine biosynthesis AICAR transformylase N10-formyl H4 folate donates formyl group to glutamine-derived amine IMP synthase cyclization of second purine ring ATP activation not required

Organization of Purine Biosynthetic Enzymes Purine biosynthesis organized into multienzyme complexes In eukaryotes, multifunctional proteins for: Steps 1, 3 & 5 Steps 6a & 8 Steps 10 & 11 In bacteria, separate enzymes associate in large complexes Channeling of intermediates avoids dilution of reactants

Synthesis of Adenylate and Guanylate AMP synthesis uses GTP for activation; amine from aspartate GMP synthesis uses ATP for activation; amide from glutamine Reciprocal Regulation: GTP for needed for AMP synthesis ATP needed for GMP synthesis

Regulation of Purine Biosynthesis in E. coli Feedback Inhibition (negative) Inhibition of 1st step in common pathway by IMP, AMP & GMP Inhibition of 1st step in branch AMP inhibits AMP synthesis GMP inhibits GMP synthesis Inhibition of PRPP synthesis by phosphorylated end products ADP, GDP and others Reciprocal Regulation (positive) Requirements of: ATP for GMP synthesis GTP for AMP synthesis

Nucleotide Biosynthesis Purine Biosynthesis Hypoxanthine (a purine) is assembled on the ribose 5-phosphate → Inosinate (IMP) Precursors: PRPP Glycine H4 folate-formate (2) HCO3– Glutamine (amide-N) (2) Aspartate (amino-N) IMP → AMP IMP → XMP → GMP Pyrimidine Biosynthesis Orotate (a pyrimidine) is made first then added to ribose 5-phosphate → Orotidylate Precursors: Carbamoyl phosphate HCO3– Glutamine (amide-N) Aspartate PRPP Orotidylate → UMP → UDP → UTP → CTP

Pyrimidine Biosynthesis Carbamoyl Phosphate Synthetase II cytosolic CPS II enzyme involved in pyrimidine biosynthesis mitochondrial CPS I involved in arginine & urea synthesis bacteria have single enzyme for both functions Steps: bicarbonate phosphate synthesis (1st activation) carbamate synthesis (NH3 from glutamine hydrolysis) carbamoyl phosphate synthesis (2nd activation)

Carbamoyl Phosphate Synthetase Bacterial enzyme has 2 subunits (blue & grey) with 3 active sites joined by a substrate channel (yellow wire mesh) 1st site: Glutamine releases NH4+ (glutamine in green) 2nd site: HCO3– is phosphorylated with ATP and reacts with NH4+ to form carbamate (ADP in blue) 3rd site: Carbamoyl phosphate is synthesized by phosphorylating carbamate with ATP (ADP in red)

Pyrimidine Biosynthesis aspartate transcarbamoylase activated carbamoyl group transferred to amine group of aspartate Pi displaced; amide bond formed committed step in pyrimidine synthesis dihydroorotase cyclization of pyrimidine ring dihydroorotate dehydrogenase oxidation of C-C bond using NAD+ orotate phosphoribosyl transferase pyrimidine ring (orotate) is transferred to activated 5-phosphoribose (PRPP) PPi lost; aminoglycan bond formed analogous to pyrimidine salvage

Pyrimidine Biosynthesis orotidylate decarboxylase catalyzes synthesis of UMP very efficient enzyme uridylate kinase nucleoside monophosphate kinase specific for UMP nucleoside diphosphate kinase generic enzyme for (d)NDP’s cytidylate synthetase an amidotransferase UTP is aminated using glutamine carbonyl group is activated with ATP to form acyl phosphate intermediate Cytidine 5’-triphosphate (CTP)

Pyrimidine Biosynthesis Enzyme Complexes Eukaryotes have a multifunctional protein with the first 3 enzymes in pyrimidine biosynthetic pathway C carbamoyl phosphate synthetase II A aspartate transcarbamoylase D dihydroorotase CAD has 3 identical polypeptides (Mr 230,000) each with sites for all 3 reactions

Regulation of Pyrimidine Biosynthesis Feedback inhibition of 1st step aspartate transcarbamoylase (ATCase) by CTP Bacterial ATCase has: 6 catalytic subunits 6 regulatory subunits Allosteric inhibition: 2 conformations of ATCase: active ↔ inactive binding of CTP to regulatory subunits shifts conformation active → inactive ATP reverses effect of CTP

Activation of Nucleotides Nucleoside monophosphate kinases specific enzyme for each base (e.g. adenylate kinase) nonspecific for ribose (ribose or 2’-deoxyribose) ATP + NMP ADP + NDP Nucleoside diphosphate kinase generic enzyme, nonspecific for base or ribose nonspecific for phosphate donor or acceptor NTP + NDP NDP + NTP donor acceptor acceptor donor

Nucleotides for DNA Synthesis 2 Modifications: ribonucleotides reduced to 2’-deoxyribonucleotides NDP → dNDP uracil (uridylate) methylated to thymine (thymidylate) dUMP → dTMP

Reduction of Nucleotides NDP is reduced to dNDP by reduced form of ribonucleotide reductase Oxidized form of ribonucleotide reductase is reduced by either glutaredoxin or thioredoxin Oxidized form of glutaredoxin is reduced by glutathione Oxidized form of thioredoxin is reduced by FADH2 Oxidized glutathione and FAD are reduced by NADPH

Regulation of Ribonucleotide Reductase Ribonucleotide Reductase (E. coli) Active sites are between each R1 and R2 subunit Two R2 subunits each contain a tyrosyl radical and a binuclear Fe3+ cofactor Two R1 subunits each have sites for enzyme activity and substrate specificity The (d)NTP bound to substrate specificity sites determines which NDP is reduced to dNDP

Regulation of Ribonucleotide Reductase Binding at activity regulatory sites: ATP activates enzyme dATP inhibits enzyme Binding at substrate specificity sites: ATP or dATP: ↑dCDP ↑dUDP dTTP: ↑dGDP ↓dCDP ↓dUDP dGTP: ↑dADP ↓dGDP ↓dCDP ↓dUDP

Biosynthesis of Thymidylate Precursors for thymidylate (dTMP) synthesis may arise from (d)CTP or (d)UTP pools CTP UTP nucleoside diphosphate kinase UMP cytidylate synthetase uridylate kinase

Cyclic pathway for conversion of dUMP to dTTP Thymidylate synthase uses N5,N10-Methylene-H4 folate as both one-carbon source and reducing agent Dihydrofolate reductase reduces H2 folate → H4 folate with NADPH Serine hydroxymethyl transferase reaction restores N5,N10-Methylene-H4 folate Net reaction: dUMP + NADPH + serine → dTMP + NADP+ + glycine

Chemotherapeutic Agents Inhibitors of glutamine amidotransferases: Block purine & pyrimidine biosynthesis Inhibitors of thymidylate synthesis: thymidylate synthase dihydrofolate reductase

Chemotherapy Targets

Group Study Problem Conversion of dUTP to dTTP by thymidylate synthase requires N5,N10-Methylene-H4 folate as both one-carbon source and reducing agent N5,N10-Methylene-H4 folate and glycine are produced in a reversible reaction whereby the hydroxymethyl group of serine in transferred to H4 folate What effect may an elevated glycine:serine ratio during photorespiration have on DNA synthesis? January 31, 2008

Catabolism of Purine Nucleotides Adenosine deaminase deficiency: severe immunodeficiency disease; loss of T- and B-cells 100x ↑ dATP (inhibitor of ribonucleotide reductase) ↓ dNTP’s, ↓ DNA synthesis Catabolism produces purine bases for salvage pathways Uric acid catabolic end product in humans gout – accumulation of uric acid in joints and urine treatment with xanthine oxidase inhibitors, e.g. allopurinol

Purine Catabolism Pyrimidine Catabolism

Salvage Pathways for Nucleotides de novo biosynthesis of purine nucleotides assembles the purine ring on 5’-phosphoribose Salvage pathway adds completed purine base to PRPP Adenosine phosphoribosyltransferase Adenine + PRPP → AMP + PPi Hypoxanthine-guanine phosphoribosyltransferase Hypoxanthine + PRPP → IMP + PPi Guanine + PRPP → GMP + PPi Lesch-Nyhan syndrome: deficiency in hypoxanthine-guanine phosphoribosyltransferase

Biosynthesis of Cofactors Nicotinamide Adenine Dinucleotide (NAD) PRPP PPi ATP PPi Gln Glu Nicotinate (Niacin) Nicotinate ribonucleotide Desamido NAD+ NAD+ Flavin Adenine Dinucleotide (FAD) ADP ATP ATP PPi Riboflavin 5’-phosphate Riboflavin FAD