Nucleic Acids Metabolism

Nitrogenous Bases Planar, aromatic, and heterocyclic Derived from purine or pyrimidine Numbering of bases is “unprimed”

Nucleic Acid Bases Pyrimidines Purines

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 N-glycosidic 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: Nucleotides: 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

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 a to b (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, or adenosinyl group ATP hydrolysis can drive an otherwise unfavorable reaction (synthetase)

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 + PRPP  AMP + PPi 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 : HCO3- N3 : Glutamine amide Nitrogen

Pyrimidine Synthesis

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 b-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