Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis
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Andújar Sánchez, Montserrat; Martínez Rodríguez, Sergio; Las Heras Vázquez, Francisco Javier; Clemente Jiménez, Josefa María; Rodríguez Vico, Felipe; [et al.]Date
2006Abstract
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, ΔG, ΔH and ΔS 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 bin...
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Binding
Mutagenesis
hydantoin racemase
Inhibition
Thermodynamic parameter