title: Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation.
abstract: Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, 6681/92-A Av Ipiranga, 90619-900 Porto Alegre, RS, Brazil.
authors.forename: Leonardo Krás Borges
authors.initials: LK
authors.lastname: Martinelli
authors.forename: Rodrigo Gay
authors.initials: RG
authors.lastname: Ducati
authors.forename: Leonardo Astolfi
authors.initials: LA
authors.lastname: Rosado
authors.forename: Ardala
authors.initials: A
authors.lastname: Breda
authors.forename: Bruna Pelegrim
authors.initials: BP
authors.lastname: Selbach
authors.forename: Diógenes Santiago
authors.initials: DS
authors.lastname: Santos
authors.forename: Luiz Augusto
authors.initials: LA
authors.lastname: Basso
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D050899:GMP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D050899:GMP Reductase