Kinetic Studies of Wheat Carboxypeptidase-Catalyzed Reaction: Differences in Pressure and Temperature Dependence of Peptidase and Esterase Activities

抄録

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3- (2-furyl) acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (k_cat/K_m) was at pH 3.3 and the k_cat/K_m value decreased with increasing pH with an apparent pK_a of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pK_a of 6.04. Optimum pH's for k_cat for the peptidase and esterase reactions were also very different and their apparent pK_a values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of k_cat and K_m, the activation volumes (ΔV^≠) and reaction volumes (ΔV), respectively, were determined. ΔV^≠ for k_cat was - 7 to - 8 ml/mol for peptidase and - 2 to - 3ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site (s).