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Nucleic Acids Metabolism Nitrogenous Bases • Planar, aromatic, and heterocyclic • Derived from purine or pyrimidine • Numbering of bases is “unprimed” Nucleic Acid Bases Purines Pyrimidines Sugars • Pentoses (5-C sugars) • Numbering of sugars is “primed” Sugars D-Ribose and 2’-Deoxyribose *Lacks a 2’-OH group Nucleosides • Result from linking one of the sugars with a purine or pyrimidine base through an Nglycosidic linkage – Purines bond to the C1’ carbon of the sugar at their N9 atoms – Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms Nucleosides Phosphate Groups • Mono-, di- or triphosphates • Phosphates can be bonded to either C3 or C5 atoms of the sugar Nucleotides • Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification Nucleotides • RNA (ribonucleic acid) is a polymer of ribonucleotides • DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides • Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine – Ribonucleotides contain Uracil – Deoxyribonucleotides contain Thymine Nucleotides • Monomers for nucleic acid polymers • Nucleoside Triphosphates are important energy carriers (ATP, GTP) • Important components of coenzymes – FAD, NAD+ and Coenzyme A Naming Conventions • Nucleosides: – Purine nucleosides end in “-sine” • Adenosine, Guanosine – Pyrimidine nucleosides end in “-dine” • Thymidine, Cytidine, Uridine • Nucleotides: – Start with the nucleoside name from above and add “mono-”, “di-”, or “triphosphate” • Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate Nucleotide Metabolism • PURINE RIBONUCLEOTIDES: formed de novo – i.e., purines are not initially synthesized as free bases – First purine derivative formed is Inosine Mono-phosphate (IMP) • The purine base is hypoxanthine • AMP and GMP are formed from IMP Purine Nucleotides • Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine ring components came from: N1: Aspartate Amine C2, C8: Formate N3, N9: Glutamine C4, C5, N7: Glycine C6: Bicarbonate Ion Purine Nucleotide Synthesis O COO OOC 2- O3P O CH2 H O H H H C OH OH OH O3P O CH2 CH 5 ADP + Pi HC N H SAICAR Synthetase CH2 C COO AIR Car boxylase ADP + Pi H H H OH OH H O Ribose-5-Phosphate Fumarate O P O O P O C O C H2N CH 5 N C 5-Aminoimidazole Ribotide (AIR) ADP + Pi N10-Formyl- H2C CH NH2 O C H H OH OH HN H C O C ADP + Pi O3P O CH2 H NH2 Ribose-5-Phosphate 5-Formaminoimidazole-4-carboxamide ribotide (FAICAR) ATP + Glutamine + H2O H2C NH O C N10-Formyl-THF THF O IMP Cyclohydrolase O C OH Glycinamide Ribotide (GAR) GAR Transformylase N CH HN C O HC C5 NH H H N NH H2O H N CH 5 C H OH ADP + Glutamate + Pi FGAM Synthetase GAR Synthetase O C H Ribose-5-Phosphate Formylglycinamidine ribotide (FGAM) N C4 NH O Glycine + ATP H 2C AICAR Transformylase THF H2N H -5-Phosphoribosylamine (PRA) 2- THF O H N Glutamate + PPi O3P O CH2 5-Aminoimidazole-4-carboxamide ribotide (AICAR) ATP Transferase 2- N Ribose-5-Phosphate AIR Synthetase Glutamine + H2O Amidophosphoribosyl CH 5 H2N Ribose-5-Phosphate 5-Phosphoribosyl--pyrophosphate (PRPP) N C4 H2N O Adenylosuccinate Lyase O N HC 4 N 5-Aminoimidazole-4-(N-succinylocarboxamide) ribotide (SAICAR) ATP +HCO3 O CH 5 H2N Ribose-5-Phosphate Ribose Phosphate Pyrophosphokinase N C4 N Carboxyamidoimidazole Ribotide (CAIR) AMP 2- Aspartate + ATP H2N -D-Ribose-5-Phosphate (R5P) ATP C N C4 Ribose-5-Phosphate Formylglycinamide ribotide (FGAR) 4 CH N N 2- O3P O CH2 H H OH O H H OH Inosine Monophosphate (IMP) Purine Nucleotide Synthesis at a Glance • ATP is involved in 6 steps • PRPP in the first step of Purine synthesis is also a precursor for Pyrimidine Synthesis, His and Trp synthesis – Role of ATP in first step is unique– group transfer rather than coupling • In second step, C1 notation changes from to (anomers specifying OH positioning on C1 with respect to C4 group) • In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step) Coupling of Reactions • Hydrolyzing a phosphate from ATP is relatively easy G°’= -30.5 kJ/mol – If exergonic reaction released energy into cell as heat energy, wouldn’t be useful – Must be coupled to an endergonic reaction • When ATP is a reactant: – Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl, adenosinyl group – ATP hydrolysis can drive an otherwise unfavorable reaction (synthetase) or Purine Biosynthetic Pathway • Channeling of some reactions on pathway organizes and controls processing of substrates to products in each step – Increases overall rate of pathway and protects intermediates from degradation • In animals, IMP synthesis pathway shows channeling at: – Reactions 3, 4, 6 – Reactions 7, 8 – Reactions 10, 11 IMP Conversion to AMP IMP Conversion to GMP Regulatory Control of Purine Nucleotide Biosynthesis • GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of production) • PRPP is a biosynthetically “central” molecule (why?) – ADP/GDP levels – negative feedback on Ribose Phosphate Pyrophosphokinase – Amidophosphoribosyl transferase is activated by PRPP levels – APRT activity has negative feedback at two sites • ATP, ADP, AMP bound at one site • GTP,GDP AND GMP bound at the other site • Rate of AMP production increases with increasing concentrations of GTP; rate of GMP production increases with increasing concentrations of ATP Regulatory Control of Purine Biosynthesis • Above the level of IMP production: – Independent control – Synergistic control – Feedforward activation by PRPP • Below level of IMP production – Reciprocal control • Total amounts of purine nucleotides controlled • Relative amounts of ATP, GTP controlled Purine Catabolism and Salvage • All purine degradation leads to uric acid • Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine • Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides – Direct absorption of nucleosides – Further degradation Nucleoside + H2O base + ribose (nucleosidase) Nucleoside + Pi base + r-1-phosphate (n. phosphorylase) NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED. Intracellular Purine Catabolism • Nucleotides broken into nucleosides by action of 5’nucleotidase (hydrolysis reactions) • Purine nucleoside phosphorylase (PNP) – – – – Inosine Hypoxanthine Xanthosine Xanthine Guanosine Guanine Ribose-1-phosphate splits off • Can be isomerized to ribose-5-phosphate • Adenosine is deaminated to Inosine (ADA) Intracellular Purine Catabolism • Xanthine is the point of convergence for the metabolism of the purine bases • Xanthine Uric acid – Xanthine oxidase catalyzes two reactions • Purine ribonucleotide degradation pathway is same for purine deoxyribonucleotides Adenosine Degradation Xanthosine Degradation • Ribose sugar gets recycled (Ribose-1-Phosphate R-5-P ) – can be incorporated into PRPP (efficiency) • Hypoxanthine is converted to Xanthine by Xanthine Oxidase • Guanine is converted to Xanthine by Guanine Deaminase • Xanthine gets converted to Uric Acid by Xanthine Oxidase Xanthine Oxidase • A homodimeric protein • Contains electron transfer proteins – FAD – Mo-pterin complex in +4 or +6 state – Two 2Fe-2S clusters • Transfers electrons to O2 H2O2 – H2O2 is toxic – Disproportionated to H2O and O2 by catalase THE PURINE NUCLEOTIDE CYCLE AMP + H2O IMP + NH4+ (AMP Deaminase) IMP + Aspartate + GTP AMP + Fumarate + GDP + Pi (Adenylosuccinate Synthetase) COMBINE THE TWO REACTIONS: Aspartate + H2O + GTP Fumarate + GDP + Pi + NH4+ The overall result of combining reactions is deamination of Aspartate to Fumarate at the expense of a GTP Uric Acid Excretion • Humans – excreted into urine as insoluble crystals • Birds, terrestrial reptiles, some insects – excrete insoluble crystals in paste form – Excess amino N converted to uric acid • (conserves water) • Others – further modification : Uric Acid Allantoin Allantoic Acid Urea Ammonia Purine Salvage • Adenine phosphoribosyl transferase (APRT) Adenine + PRPP AMP + PPi • Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT) Hypoxanthine + PRPP IMP + PPi Guanine + PRPP GMP + PPi (NOTE: THESE ARE ALL REVERSIBLE REACTIONS) AMP,IMP,GMP do not need to be resynthesized de novo ! Pyrimidine Ribonucleotide Synthesis • Uridine Monophosphate (UMP) is synthesized first – CTP is synthesized from UMP • Pyrimidine ring synthesis completed first; then attached to ribose-5-phosphate N1, C4, C5, C6 : Aspartate C2 : HCO3N3 : Glutamine amide Nitrogen Pyrimidine Synthesis O 2 ATP + HCO3- + Glutamine + H2O C 2 ADP + Glutamate + Pi O Carbamoyl Phosphate Synthetase II C C NH2 CH C N H PO3-2 O PRPP C O C C N O HN O CH HN PPi 2- COO O3P O Orotate Phosphoribosyl Transferase CH2 O H H OH OH H H COO Orotidine-5'-monophosphate (OMP) Orotate Carbamoyl Phosphate Aspartate Reduced Quinone Aspartate Transcarbamoylase (ATCase) O C CH2 CH N H O 2- CH N H COO Dihydroorotate COO O3P O CH2 CH N O H2O Dihydroorotase Carbamoyl Aspartate C CH2 HN C O CH HN C C C O O NH2 CO2 Quinone Pi HO OMP Decarboxylase Dihydroorotate Dehydrogenase O H H OH OH H H Uridine Monophosphate (UMP) UMP Synthesis Overview • 2 ATPs needed: both used in first step – One transfers phosphate, the other is hydrolyzed to ADP and Pi • 2 condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular) • Dihydroorotate dehydrogenase is an intra-mitochondrial enzyme; oxidizing power comes from quinone reduction • Attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-P – PPi splits off PRPP – irreversible UMP UTP and CTP • Nucleoside monophosphate kinase catalyzes transfer of Pi to UMP to form UDP; nucleoside diphosphate kinase catalyzes transfer of Pi from ATP to UDP to form UTP • CTP formed from UTP via CTP Synthetase driven by ATP hydrolysis – Glutamine provides amide nitrogen for C4 in animals Regulatory Control of Pyrimidine Synthesis • Differs between bacteria and animals – Bacteria – regulation at ATCase rxn • Animals – regulation at carbamoyl phosphate synthetase II – UDP and UTP inhibit enzyme; ATP and PRPP activate it – UMP and CMP competitively inhibit OMP Decarboxylase *Purine synthesis inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, controlling level of PRPP also regulates pyrimidines Degradation of Pyrimidines • CMP and UMP degraded to bases similarly to purines – Dephosphorylation – Deamination – Glycosidic bond cleavage • Uracil reduced in liver, forming -alanine – Converted to malonyl-CoA fatty acid synthesis for energy metabolism Deoxyribonucleotide Formation • Purine/Pyrimidine degradation are the same for ribonucleotides and deoxyribonucleotides • Biosynthetic pathways are only for ribonucleotide production • Deoxyribonucleotides are synthesized from corresponding ribonucleotides DNA vs. RNA: REVIEW • DNA composed of deoxyribonucleotides • Ribose sugar in DNA lacks hydroxyl group at 2’ Carbon • Uracil doesn’t (normally) appear in DNA – Thymine (5-methyluracil) appears instead Formation of Deoxyribonucleotides • Reduction of 2’ carbon done via a free radical mechanism catalyzed by “Ribonucleotide Reductases” – E. coli RNR reduces ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs) • Two subunits: R1 and R2 – A Heterotetramer: (R1)2 and (R2)2 Thymine Formation • Formed by methylating deoxyuridine monophosphate (dUMP) • UTP is needed for RNA production, but dUTP not needed for DNA – If dUTP produced excessively, would cause substitution errors (dUTP for dTTP) • dUTP hydrolyzed by dUTPase (dUTP diphosphohydrolase) to dUMP methylated at C5 to form dTMP rephosphorylate to form dTTP