Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis.
Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, deltaG, deltaH and DeltaS for the inhibitors L- and D-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.
Andújar-Sánchez
Montserrat
M
Dpto. Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Carretera Sacramento s/n Almería, 04120, España.
Martínez-Rodríguez
Sergio
S
Heras-Vázquez
Francisco Javier Las
FJ
Clemente-Jiménez
Josefa María
JM
Rodríguez-Vico
Felipe
F
Jara-Pérez
Vicente
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D008970:Molecular Weight; D009154:Mutation; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D016962:Sinorhizobium meliloti / Q000201:enzymology*; D013816:Thermodynamics
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D019998:Racemases and Epimerases; C074773:hydantoin racemase; D003545:Cysteine