06 May 2008 Nucleic Acid Metabolism Andy Howard Introductory Biochemistry 6 May 2008

06 May 2008 Nucleic Acid Metabolism p.2 of 56 What we’ll discuss Pyrimidine synthesis PRPP Pathway to UMP Regulation Pathway to CTP Purine synthesis IMP AMP, XMP, GMP Regulation Reduction of riboNucs to deoxyNucs dUMP to dTMP Salvage pathways Pyrimidine catabolism Purine catabolism

06 May 2008 Nucleic Acid Metabolism p.3 of 56 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 Phosphoribosyl pyrophosphate PRPP synthetase PDB 2H kDa hexamer dimer shown; human

06 May 2008 Nucleic Acid Metabolism p.4 of 56 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 + P i via aspartate transcarbamoylase Carbamoyl aspartate Carbamoyl phosphate

06 May 2008 Nucleic Acid Metabolism p.5 of 56 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 ATCase PDB 1D09 Trimer of heterotetramers 1 heterotetramer shown (cf. fig.18.11) E.coli

06 May 2008 Nucleic Acid Metabolism p.6 of 56 Carbamoyl aspartate to dihydroorotate Carbamoyl aspartate dehydrates and cyclizes to L- dihydroorotate via dihydroorotase TIM barrel protein Dihydro- orotate PDB 1XGE 76 kDa dimer E.coli

06 May 2008 Nucleic Acid Metabolism p.7 of 56 Dihydroorotate to orotate Ubiquinone acts as oxidizing agent reducing the 5 & 6 Carbons via dihydroorotate dehydrogenase Some versions incorporate FMN PDB 2E6F 69 kDa dimer Trypanosoma cruzi

06 May 2008 Nucleic Acid Metabolism p.8 of 56 Adding phosphoribose Orotate + PRPP  orotidine 5’- monophosphate + PP i Usual argument re pyrophosphate hydrolysis Enzyme: orotidine phosphoribosyl transferase Orotidine 5’- monophosphate PDB 2PS1 50 kDa dimer Yeast

06 May 2008 Nucleic Acid Metabolism p.9 of 56 Decarboxylation OMP decarboxylated to form UMP via OMP decarboxylase Bacterial forms are TIM barrel proteins Acceleration is fold relative to uncatalyzed rate PDB 1KLY 54 kDa dimer Methanobacterium thermoautotrophicum

06 May 2008 Nucleic Acid Metabolism p.10 of 56 Eukaryotic variation Orotate produced in the mitochondrion moves to the cytosol UMP synthase combines the last two reactions—orotidine to OMP to UMP OMP decarboxylase domain of UMP synthase PDB 2P1F 64 kDa dimer human

06 May 2008 Nucleic Acid Metabolism p.11 of 56 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) Uridylate kinase PDB 2A1F 163 kDa hexamer Haemophilus influenzae

06 May 2008 Nucleic Acid Metabolism p.12 of 56 CTP synthetase UTP + gln + ATP  CTP + glu + ADP + P i 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!) PDB 1S1M 240 kDa tetramer dimer shown E.coli

06 May 2008 Nucleic Acid Metabolism p.13 of 56 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

06 May 2008 Nucleic Acid Metabolism p.14 of 56 PRPP + gln to phospho- ribosylamine PRPP aminated: PRPP + gln  glu + PP i + 5-phospho-  -D- ribosylamine via glutamine-PRPP amidotransferase  transferase structure Product is unstable (lasts seconds!) 1 PDB 1ECF 120 kDa tetramer dimer shown E.coli

06 May 2008 Nucleic Acid Metabolism p.15 of 56 Phospho- ribosylamine to GAR 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 2

06 May 2008 Nucleic Acid Metabolism p.16 of 56 Formylation of GAR 10-formyl THF donates a formyl (-CH=O) group to end nitrogen with the help of GAR transformylase to form formylglycinamide ribonucleotide (FGAR) Rossmann  FGAR PDB 1MEO 47 kDa dimer human 3

06 May 2008 Nucleic Acid Metabolism p.17 of 56 FGAR to FGAM Glutamine sidechain is source of N for C=O exchanging to C=NH via FGAM synthetase to form formylglycinamidine ribonucleotide (FGAM): FGAR + gln + ATP + H 2 O  FGAM + glu + ADP + P i PurS component of FGAM synthetase PDB 1GTD 37.4 kDa tetramer dimer shown Methanobacterium FGAM 4

06 May 2008 Nucleic Acid Metabolism p.18 of 56 FGAM to AIR Cyclize FGAM to aminoimidazole ribonucleotide ATP drives the AIR synthetase reaction: FGAM + ATP  AIR + H 2 O + ADP + P i E.C. in Wikipedia is wrong: it should be PDB 2V9Y 147 kDa tetramer dimer shown human Aminoimidazole ribonucleotide 5

06 May 2008 Nucleic Acid Metabolism p.19 of 56 AIR to CAIR AIR is carboxylated; expenditure of an ATP: AIR + HCO ATP  carboxyaminoimidazole ribonucleotide + ADP + P i + 2H + AIR carboxylase E.coli version is two enzymes; eukaryotes have a single enzyme No cofactors! PDB 2NSH 149 kDa octamer monomer shown E.coli CAIR 6

06 May 2008 Nucleic Acid Metabolism p.20 of 56 CAIR+asp to SAICAR CAIR + asp + ATP  aminoimidazole succinylocarboxamide ribonucleotide + ADP + P i Enzyme is SAICAR synthetase Domain 1: homolog of phosphorylase kinase Domain 2: ATP-binding PDB 2CNQ 34 kDa monomer yeast 7

06 May 2008 Nucleic Acid Metabolism p.21 of 56 SAICAR to AICAR 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 PDB 2PTR 203 kDa tetramer dimer shown; E.coli 8

06 May 2008 Nucleic Acid Metabolism p.22 of 56 AICAR to FAICAR 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) 9 PDB 1THZ 130 kDa dimer chicken

06 May 2008 Nucleic Acid Metabolism p.23 of 56 FAICAR to IMP We made it: FAICAR  inosine 5’- monosphosphate + H 2 O Bifunctional enzyme; this part is called IMP cyclohydrolase or inosicase Hydrolase part is like methylglyoxal synthase PDB 1PL0 260 kDa tetramer dimer shown; human 10

06 May 2008 Nucleic Acid Metabolism p.24 of 56 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

06 May 2008 Nucleic Acid Metabolism p.25 of 56 IMP to adenylosuccinate IMP + aspartate + GTP  adenylosuccinate + GDP + P i Enzyme is adenylosuccinate synthase Similar to step 7 in IMP synthesis PDB 2V kDa dimer monomer shown human

06 May 2008 Nucleic Acid Metabolism p.26 of 56 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

06 May 2008 Nucleic Acid Metabolism p.27 of 56 IMP to XMP IMP + H 2 O + NAD +  Xanthosine monophosphate + NADH + H + Enzyme: IMP dehydrogenase TIM-barrel, aldolase- like protein PDB 1ME8 221 kDa tetramer; monomer shown Tritrichomonas foetus

06 May 2008 Nucleic Acid Metabolism p.28 of 56 XMP to GMP XMP + gln + H 2 O + ATP  GMP + glu + AMP + PPi Enzyme: GMP synthetase Typical 3-layer  sandwich PDB 2DPL 68 kDa dimer Pyrococcus horikoshii

06 May 2008 Nucleic Acid Metabolism p.29 of 56 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 PDB 1ZIN 24 kDa monomer Bacillus stearothermophilus

06 May 2008 Nucleic Acid Metabolism p.30 of 56 Guanylate kinase GMP + ATP  GDP + ADP “P-loop”-containing ATP- binding proteins Rossmann fold PDB 2QOR 22 kDa monomer Plasmodium vivax

06 May 2008 Nucleic Acid Metabolism p.31 of 56 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

06 May 2008 Nucleic Acid Metabolism p.32 of 56 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

06 May 2008 Nucleic Acid Metabolism p.33 of 56 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

06 May 2008 Nucleic Acid Metabolism p.34 of 56 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 2 RNR2 subunits; each has A diferric ion center A stable tyrosyl free radical RNR1 PDB 1R1R 258 kDa dimer E.coli RNR2 PDB 1PJ0 82 kDa dimer E.coli

06 May 2008 Nucleic Acid Metabolism p.35 of 56 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

06 May 2008 Nucleic Acid Metabolism p.36 of 56 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

06 May 2008 Nucleic Acid Metabolism p.37 of 56 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 + H 2 O  dUMP + PP i dCMP + H 2 O  dUMP + NH 4 +

06 May 2008 Nucleic Acid Metabolism p.38 of 56 Thymidylate synthase reaction (fig.18.15) dUMP + 5,10-methyleneTHF  dTMP + 7,8-dihydrofolate Unusual THF reaction in that cofactor gets oxidized as well as giving up a carbon CH 2 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 5,10-methylene THF dihydrofolate

06 May 2008 Nucleic Acid Metabolism p.39 of 56 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 PDB 2G8O 58 kDa dimer E.coli (with dUMP and cofactor analog)

06 May 2008 Nucleic Acid Metabolism p.40 of 56 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

06 May 2008 Nucleic Acid Metabolism p.41 of 56 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 PDB 1KMV 20 kDa monomer human folate

06 May 2008 Nucleic Acid Metabolism p.42 of 56 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: DHFR-TS PDB 1J3K 104 kDa dimer Plasmodium falciparum

06 May 2008 Nucleic Acid Metabolism p.43 of 56 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 PDB 1E2K 73 kDa monomer Herpes simplex virus

06 May 2008 Nucleic Acid Metabolism p.44 of 56 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

06 May 2008 Nucleic Acid Metabolism p.45 of 56 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

06 May 2008 Nucleic Acid Metabolism p.46 of 56 Orotate phosphoribosyl transferase Principal salvage enzyme for pyrimidines Orotate + PRPP -> OMP + PP i 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 + PP i PDB 2 PS1 50 kDa dimer Yeast

06 May 2008 Nucleic Acid Metabolism p.47 of 56 Pyrimidine interconversions (fig ) 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

06 May 2008 Nucleic Acid Metabolism p.48 of 56 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 Hypoxanthine- guanine phosphoribosyl transferase PDB 1FSG 102 kDa tetramer dimer shown Toxoplasma gondii

06 May 2008 Nucleic Acid Metabolism p.49 of 56 Purine Interconnections (fig ) 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

06 May 2008 Nucleic Acid Metabolism p.50 of 56 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 Cytidine deaminase PDB 2FR5 64 kDa tetramer Mouse

06 May 2008 Nucleic Acid Metabolism p.51 of 56 Hydrolysis of U, dU and dT Glycosidic bond in uridine or thymidine is hydrolyzed by phosphate: Uridine + P i ->  -D-ribose-1-P + uracil Enyzme is uridine phosphorylase Similar enzyme handles deoxyuridine Similar reaction using thymidine phosphorylase yields thymine +  -D- deoxyribose-1-P Uridine phosphorylase PDB 1RXY 167 kDa hexamer Dimer shown E.coli

06 May 2008 Nucleic Acid Metabolism p.52 of 56 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 Dihydro- pyrimidinase PDB 1GKP 302 kDa hexamer Thermus

06 May 2008 Nucleic Acid Metabolism p.53 of 56 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 Uric acid

06 May 2008 Nucleic Acid Metabolism p.54 of 56 Uric acid to allantoin Urate oxidase: urate + 2H 2 O + O 2  allantoin + H 2 O 2 + CO 2 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 Uric acid Allantoin Urate oxidase 134 kDa tetramer monomer shown Aspergillus flavus

06 May 2008 Nucleic Acid Metabolism p.55 of 56 Lesch-Nyhan syndrome 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 Michael Lesch William Nyhan

06 May 2008 Nucleic Acid Metabolism p.56 of 56 Gout 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 Sodium urate crystals accumulating Sodium urate Benjamin Franklin (celebrated gout sufferer)