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Nucleic Acid Metabolism Andy Howard Introductory Biochemistry 6 May 2008 Nucleic Acid Metabolism 06 May 2008 What we’ll discuss Pyrimidine synthesis PRPP Pathway to UMP Regulation Pathway to CTP Purine synthesis IMP AMP, XMP, GMP Regulation Nucleic Acid Metabolism Reduction of riboNucs to deoxyNucs dUMP to dTMP Salvage pathways Pyrimidine catabolism Purine catabolism p.2 of 56 06 May 2008 Phosphoribosyl pyrophosphate PRPP synthetase Activation of ribose-5-P (see Calvin cycle, etc.) by ATP: -ribose-5-P + ATP PRPP + AMP Has roles in other systems too Nucleic Acid Metabolism PRPP synthetase PDB 2H06 215 kDa hexamer dimer shown; human p.3 of 56 06 May 2008 Pyrimidine synthesis: carbamoyl aspartate Uridine is based on orotate, which is derivated from carbamoyl aspartate We’ve already seen the carbamoyl phosphate synthesis back in chapter 17 via carbamoyl phosphate synthetase Carbamoyl phosphate + aspartate carbamoyl aspartate + Pi via aspartate transcarbamoylase Nucleic Acid Metabolism p.4 of 56 Carbamoyl phosphate Carbamoyl aspartate 06 May 2008 Aspartate transcarbamoylase ATCase is the classic allosteric enzyme E.coli version is inhibited by pyrimidine nucleotides and activated by ATP CTP by itself is 50% inhibitory; CTP+ UTP is almost totally inhibitory Nucleic Acid Metabolism p.5 of 56 ATCase PDB 1D09 Trimer of heterotetramers 1 heterotetramer shown (cf. fig.18.11) E.coli 06 May 2008 Carbamoyl aspartate to dihydroorotate Dihydroorotate Carbamoyl aspartate dehydrates and cyclizes to Ldihydroorotate via dihydroorotase TIM barrel protein Nucleic Acid Metabolism p.6 of 56 PDB 1XGE 76 kDa dimer E.coli 06 May 2008 Dihydroorotate to orotate Ubiquinone acts as oxidizing agent reducing the 5 & 6 Carbons via dihydroorotate dehydrogenase Some versions incorporate FMN Nucleic Acid Metabolism p.7 of 56 PDB 2E6F 69 kDa dimer Trypanosoma cruzi 06 May 2008 Adding phosphoribose Orotidine 5’monophosphate Orotate + PRPP orotidine 5’monophosphate + PPi Usual argument re pyrophosphate hydrolysis Enzyme: orotidine phosphoribosyl transferase Nucleic Acid Metabolism p.8 of 56 PDB 2PS1 50 kDa dimer Yeast 06 May 2008 Decarboxylation OMP decarboxylated to form UMP via OMP decarboxylase Bacterial forms are TIM barrel proteins Acceleration is 1017-fold relative to uncatalyzed rate Nucleic Acid Metabolism p.9 of 56 PDB 1KLY 54 kDa dimer Methanobacterium thermoautotrophicum 06 May 2008 Eukaryotic variation Orotate produced in the mitochondrion moves to the cytosol UMP synthase combines the last two reactions—orotidine to OMP to UMP Nucleic Acid Metabolism p.10 of 56 OMP decarboxylase domain of UMP synthase PDB 2P1F 64 kDa dimer human 06 May 2008 UMP to UTP Uridylate kinase converts UMP to UDP: UMP + ATP UDP + ADP enzyme is related to several amino acid kinases Nucleoside diphosphate kinase exchanges di for tri: UDP + ATP UTP +ADP (non-specific enzyme) Nucleic Acid Metabolism p.11 of 56 Uridylate kinase PDB 2A1F 163 kDa hexamer Haemophilus influenzae 06 May 2008 CTP synthetase UTP + gln + ATP CTP + glu + ADP + Pi Glutamine side-chain is amine donor ATP provides energy sandwich (Rossmann) Enzyme is inhibited by CTP In E.coli, it’s activated by GTP (makes sense!) Nucleic Acid Metabolism p.12 of 56 PDB 1S1M 240 kDa tetramer dimer shown E.coli 06 May 2008 Purine synthesis Considerably more complex than pyrimidine synthesis More atoms to condense and two rings to make More ATP to sacrifice during synthesis Several synthetase (ligase) reactions require ATP Based on PRPP, gln, 10-formyl THF, asp Nucleic Acid Metabolism p.13 of 56 06 May 2008 PRPP + gln to phosphoribosylamine 1 PRPP aminated: PRPP + gln glu + PPi + 5-phospho--Dribosylamine via glutamine-PRPP amidotransferase transferase structure Product is unstable (lasts seconds!) Nucleic Acid Metabolism p.14 of 56 PDB 1ECF 120 kDa tetramer dimer shown E.coli 06 May 2008 Phosphoribosylamine to GAR 2 Amine condenses with glycine to form glycinamide ribonucleotide (GAR) ATP hydrolysis drives GAR synthetase reaction to the right PDB 2YRX 50 kDa monomer Geobacillus kaustophilus Nucleic Acid Metabolism p.15 of 56 06 May 2008 FGAR Formylation of GAR 3 10-formyl THF donates a formyl (-CH=O) group to end nitrogen with the help of GAR transformylase to form formylglycinamide ribonucleotide (FGAR) Rossmann Nucleic Acid Metabolism p.16 of 56 PDB 1MEO 47 kDa dimer human 06 May 2008 FGAR to FGAM 4 Glutamine sidechain is source of N for C=O exchanging to C=NH via FGAM synthetase to form formylglycinamidine ribonucleotide (FGAM): FGAR + gln + ATP + H2O FGAM + glu + ADP + Pi PurS component of FGAM synthetase PDB 1GTD 37.4 kDa tetramer dimer shown Methanobacterium Nucleic Acid Metabolism p.17 of 56 FGAM 06 May 2008 Aminoimidazole ribonucleotide FGAM to AIR 5 Cyclize FGAM to aminoimidazole ribonucleotide ATP drives the AIR synthetase reaction: FGAM + ATP AIR + H2O + ADP + Pi E.C. in Wikipedia is wrong: it should be 6.3.3.1 Nucleic Acid Metabolism PDB 2V9Y 147 kDa tetramer dimer shown human p.18 of 56 06 May 2008 CAIR AIR to CAIR 6 AIR is carboxylated; expenditure of an ATP: AIR + HCO3- + ATP carboxyaminoimidazole ribonucleotide + ADP + Pi + 2H+ AIR carboxylase E.coli version is two enzymes; eukaryotes have a single enzyme No cofactors! Nucleic Acid Metabolism p.19 of 56 PDB 2NSH 149 kDa octamer monomer shown E.coli 06 May 2008 CAIR+asp to SAICAR 7 CAIR + asp + ATP aminoimidazole succinylocarboxamide ribonucleotide + ADP + Pi Enzyme is SAICAR synthetase Domain 1: homolog of phosphorylase kinase Domain 2: ATP-binding Nucleic Acid Metabolism p.20 of 56 PDB 2CNQ 34 kDa monomer yeast 06 May 2008 SAICAR to AICAR 8 SAICAR aminoimidazole carboxamide ribonucleotide + fumarate Enzyme is adenylosuccinate lyase Net result of two reactions is just replacing acid with amide; That’s like first 2 reactions in urea cycle, except ADP, not AMP, is the product Nucleic Acid Metabolism p.21 of 56 PDB 2PTR 203 kDa tetramer dimer shown; E.coli 06 May 2008 AICAR to FAICAR 9 10-formylTHF donates HC=O: AICAR + 10-formylTHF formamidoimidazole carboxamide ribonucleotide + THF Enzyme: AICAR transformylase Like step 3 Generally a bifunctional enzyme combined with next step This part is like cytidine deaminase (see below) Nucleic Acid Metabolism p.22 of 56 PDB 1THZ 130 kDa dimer chicken 06 May 2008 FAICAR to IMP 10 We made it: FAICAR inosine 5’monosphosphate + H2O Bifunctional enzyme; this part is called IMP cyclohydrolase or inosicase Hydrolase part is like methylglyoxal synthase Nucleic Acid Metabolism PDB 1PL0 260 kDa tetramer dimer shown; human p.23 of 56 06 May 2008 So now we have a purine. What next? Enzymatic conversions to AMP or GMP; Details on next few slides AMP and GMP can be further phosphorylated to make ADP, GDP with specific kinases (adenylate kinase and guanylate kinase) GTP made with broad-spectrum nucleoside diphosphate kinase Nucleic Acid Metabolism p.24 of 56 06 May 2008 IMP to adenylosuccinate IMP + aspartate + GTP adenylosuccinate + GDP + Pi Enzyme is adenylosuccinate synthase Similar to step 7 in IMP synthesis PDB 2V40 101 kDa dimer monomer shown human Nucleic Acid Metabolism p.25 of 56 06 May 2008 Adenylosuccinate to AMP Adenylosuccinate AMP + fumarate Like reaction 8 in the IMP pathway; in fact, it uses the same enzyme, adenylosuccinate lyase PDB 2PTR 203 kDa tetramer dimer shown; E.coli Nucleic Acid Metabolism p.26 of 56 06 May 2008 IMP to XMP IMP + H2O + NAD+ Xanthosine monophosphate + NADH + H+ Enzyme: IMP dehydrogenase TIM-barrel, aldolase- PDB 1ME8 like protein 221 kDa tetramer; monomer shown Tritrichomonas foetus Nucleic Acid Metabolism p.27 of 56 06 May 2008 XMP to GMP XMP + gln + H2O + ATP GMP + glu + AMP + PPi Enzyme: GMP synthetase Typical 3-layer sandwich Nucleic Acid Metabolism PDB 2DPL 68 kDa dimer Pyrococcus horikoshii p.28 of 56 06 May 2008 Adenylate kinase Reminder: ATP + AMP 2 ADP Metal ions play a role in enzyme structure Enzymes like this need to shield their active sites from water to avoid pointless hydrolysis of ATP Nucleic Acid Metabolism PDB 1ZIN 24 kDa monomer Bacillus stearothermophilus p.29 of 56 06 May 2008 Guanylate kinase GMP + ATP GDP + ADP “P-loop”-containing ATPbinding proteins Rossmann fold PDB 2QOR 22 kDa monomer Plasmodium vivax Nucleic Acid Metabolism p.30 of 56 06 May 2008 Purine control I: IMP level Note that GTP is a cosubstrate in making AMP from IMP ATP is a cosubstrate in making GMP from IMP So this helps balance the 2 products Nucleic Acid Metabolism p.31 of 56 06 May 2008 Purine control II: feedback inhibition PRPP synthetase inhibited by purines, but only at unrealistic concentrations of [Pur] Step 1 (gln-PRPP amidotransferase) is allosterically inhibited by IMP, AMP, GMP Adenylosuccinate synthetase is inhibited by AMP XMP and GMP inhibit IMP dehydrogenase Nucleic Acid Metabolism p.32 of 56 06 May 2008 Making deoxyribonucleotides Conversions of nucleotides to deoxynucleotides occurs at the diphosphate level Reichard showed that most organisms have a single ribonucleotide reductase that converts ADP, GDP, CDP, UDP to dADP, dGDP, dCDP, and dUDP NADPH is the reducing agent Nucleic Acid Metabolism p.33 of 56 06 May 2008 RNR1 PDB 1R1R 258 kDa dimer E.coli Ribonucleotide reductase heterotetramer 2 RNR1 subunits; each has a helical 220-aa domain 10-strand 480-aa structure (thiols here) 5-strand 70-aa structure RNR2 PDB 1PJ0 82 kDa dimer E.coli 2 RNR2 subunits; each has A diferric ion center A stable tyrosyl free radical Nucleic Acid Metabolism p.34 of 56 06 May 2008 Mechanism of RNR (box 18.3) Y122 in RNR2 is converted to stable free radical Radical transmitted to RNR1 cys439 Cys439 reacts with substrate 3’-OH to form free radical at C3’ Substrate dehydrates to carbonyl at C3’ and free radical at C2’; S- formed at Cys462 Disulfide formed between Cys462,Cys225; radical regenerated at Cys439 Nucleic Acid Metabolism p.35 of 56 06 May 2008 Ribonucleotide reductase: control ATP, dATP, dTTP, and dGTP act as allosteric modulators by binding to two regulatory sites on the enzyme Activity site (A) regulates activity of catalytic site When ATP binds at A, activity goes up When dATP binds at A, activity inhibited overall Specificity site (S) controls which substrates can be turned over ATP at A + ATP or dATP at S : pyrimidines only dTTP at S : activates reduction of GDP dGTP at S : activates reduction of ADP Nucleic Acid Metabolism p.36 of 56 06 May 2008 dUDP to dUMP (for making dTMP) dTMP formed at monophosphate level (from dUMP) dUMP derived three ways: dUDP + ADP dUMP + ATP dUDP + ATP dUTP + ADP dUTP + H2O dUMP + PPi dCMP + H2O dUMP + NH4+ Nucleic Acid Metabolism p.37 of 56 06 May 2008 Thymidylate synthase reaction (fig.18.15) dUMP + 5,10-methyleneTHF dTMP + 7,8-dihydrofolate dihydrofolate Unusual THF reaction in that cofactor gets oxidized as well as giving up a carbon 5,10-methylene THF CH2 from 5,10-methylene group extra H from C6 So DHF must be reduced back to THF via DHFR and get its methylene back from SHMT Nucleic Acid Metabolism p.38 of 56 06 May 2008 Thymidylate synthase Generally the controlling step in DNA synthesis because [dTTP] < other [deoxynucleoside triphosphates] Therefore a target for cancer chemotherapy and other therapies that target rapidly-dividing cells Enzyme is a 2-layer sandwich Nucleic Acid Metabolism p.39 of 56 PDB 2G8O 58 kDa dimer E.coli (with dUMP and cofactor analog) 06 May 2008 Thymidylate synthase and drug design Both folate analogs and dUMP analogs can interfere with (DHFR SHMT dTMP synthase … ) cycle 5-fluorouracil is specific to thymidylate synthase Nucleic Acid Metabolism p.40 of 56 06 May 2008 DHFR Converts DHF to THF: DHF + NADPH + H+ <-> THF + NADP+ SHMT then converts THF to 5,10-methyleneTHF 3-layer sandwich Often the target for drug design as well Eukaryotic DHFR also catalyzes folate DHF Prokaryotic DHFR doesn’t; DHF derived by another mechanism in bacteria Nucleic Acid Metabolism p.41 of 56 PDB 1KMV 20 kDa monomer human folate 06 May 2008 Special case: protozoan TSynth/DHFR Bifunctional enzyme: Thymidylate synthase Dihydrofolate reductase Presumably some entropic advantage Maybe electrostatics too, allowing the negative charges on DHF to tunnel through; but cf. Atreya et al (2003) J.Biol.Chem. 278:28901. Nucleic Acid Metabolism p.42 of 56 DHFR-TS PDB 1J3K 104 kDa dimer Plasmodium falciparum 06 May 2008 Recovery pathway to dTMP Deoxythymidine can be phosphorylated by thymidine kinase: deoxythymidine + ATP dTMP + ADP Labeled thymidine is convenient for monitoring intracellular synthesis of DNA because thymidine enters cells easily Nucleic Acid Metabolism p.43 of 56 PDB 1E2K 73 kDa monomer Herpes simplex virus 06 May 2008 Fates of polynucleotides Nucleic acids hydrolyzed to mononucleotides via nucleases Mononucleotides are dephosphorylated via nucleotidases and phosphatases Resulting nucleosides are deglycosylated via nucleosideases or nucleoside phosphorylases Resulting bases are sent either into salvage pathways or get degraded and excreted Nucleic Acid Metabolism p.44 of 56 06 May 2008 Salvage pathways We can describe them, and we will: but why do they matter so much? They provide energy savings relative to de novo synthesis (think of all the ATP we used in making IMP!) Considerable medical significance to interference with these pathways Intracellular nucleic acid bases are usually recycled; dietary bases are usually broken down and excess nitrogen excreted Nucleic Acid Metabolism p.45 of 56 06 May 2008 Orotate phosphoribosyl transferase Principal salvage enzyme for pyrimidines Orotate + PRPP -> OMP + PPi OMP can then reenter UMP synthetic pathway (decarboxylation to UMP, then form UDP and CDP) Same enzyme can aact on other pyrimidines to make nucleotides: Pyr + PRPP -> PyrMP + PPi Nucleic Acid Metabolism p.46 of 56 PDB 2 PS1 50 kDa dimer Yeast 06 May 2008 Pyrimidine interconversions (fig. 18.19) All phosphorylations & dephosphorylations can and do happen UTP can be aminated to CTP CDP and UDP can be reduced to dCDP and dUDP dCMP can deaminate to dUMP Cytidine can be converted to uridine dUMP can be methylated to dTMP Nucleic Acid Metabolism p.47 of 56 06 May 2008 Purine nucleotide salvage Two phosphoribosyl transferases convert adenine, guanine, and hypoxanthine to AMP, GMP, and IMP Adenine phosphoribosyl transferase is specific HGPRT accepts both hypoxanthine and guanine Nucleic Acid Metabolism p.48 of 56 Hypoxanthineguanine phosphoribosyl transferase PDB 1FSG 102 kDa tetramer dimer shown Toxoplasma gondii 06 May 2008 Purine Interconnections (fig. 18.18) All phosphorylations and dephosphorylations can and do occur ADP and GDP can be reduced to dADP and dGDP AMP can deaminated to IMP (new) IMP can be aminated to AMP IMP can oxidized to XMP XMP can be aminated to GMP Guanine, adenine can be phosphoribosylated to GMP and AMP Nucleic Acid Metabolism p.49 of 56 06 May 2008 Fates of CMP and cytidine CMP’s phosphate can be hydrolyzed off That’s followed by deamination of cytidine to make uridine Catalyzed by cytidine deaminase Another sandwich protein Nucleic Acid Metabolism p.50 of 56 Cytidine deaminase PDB 2FR5 64 kDa tetramer Mouse 06 May 2008 Hydrolysis of U, dU and dT Glycosidic bond in uridine or thymidine is hydrolyzed by phosphate: Uridine + Pi -> -D-ribose-1-P + uracil Enyzme is uridine phosphorylase Similar enzyme handles deoxyuridine Similar reaction using thymidine phosphorylase yields thymine + -Ddeoxyribose-1-P Nucleic Acid Metabolism p.51 of 56 Uridine phosphorylase PDB 1RXY 167 kDa hexamer Dimer shown E.coli 06 May 2008 Uracil to acetyl CoA; thymine to succinyl CoA Reduced to dihydrouracil and dihydrothymine Hydrated and ring-opened to ureidopropionate or ureidoisobutyrate Eliminate bicarbonate and ammonium to yield -alanine or aminoisobutyrate Several reactions from there to acetyl CoA and succinyl CoA Nucleic Acid Metabolism p.52 of 56 Dihydropyrimidinase PDB 1GKP 302 kDa hexamer Thermus 06 May 2008 Uric acid Purine catabolism Nucleoside or deoxynucleoside + phosphate base + (D)-ribose 1-P Hypoxanthine and guanine both lead to uric acid as a product Uric acid is the final excreted nitrogenous compound in primates and birds and some reptiles Other organisms catabolize it further Nucleic Acid Metabolism p.53 of 56 06 May 2008 Uric acid Uric acid to allantoin Urate oxidase: urate + 2H2O + O2 allantoin + H2O2 + CO2 That’s the final product in a lot of mammals, turtles, some insects, gastropods Other organisms catabolize allantoin further; we’ll talk about that on Thursday Nucleic Acid Metabolism p.54 of 56 Allantoin Urate oxidase 134 kDa tetramer monomer shown Aspergillus flavus 06 May 2008 Lesch-Nyhan syndrome Michael Lesch William Nyhan Complete lack of hypoxanthine-guanine phosphoribosyl transferase So hypoxanthine and guanine are degraded to uric acid rather than being built back up into IMP and GMP Leads to dangerous buildup of uric acid in nervous tissue Neurological effects are severe and poorly understood Nucleic Acid Metabolism p.55 of 56 06 May 2008 Sodium urate Gout Sodium urate crystals accumulating Accumulation of sodium urate and uric acid, both of which are only moderately soluble Arises from inadequate (~10%) functionality of HGPRT, so that urate accumulates in peripheral tissues, particularly the feet Nucleic Acid Metabolism p.56 of 56 Benjamin Franklin (celebrated gout sufferer) 06 May 2008