Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre.
Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.
Martínez-Rodríguez
Sergio
S
Departamento de Química-Física, Bioquímica y Química Inorgánica, Edificio C.I.T.E. I, Universidad de Almería, La Cañada de San Urbano, Almería 04120, Spain.
Andújar-Sánchez
Montserrat
M
Clemente Jiménez
Josefa María
JM
Jara-Pérez
Vicente
V
Rodríguez-Vico
Felipe
F
Las Heras-Vázquez
Francisco Javier
FJ
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
D050505:Mutant Proteins; D000581:Amidohydrolases; C451299:N-carbamoylamino acid amidohydrolase