PH DEPENDENCE AND COMPETITIVE PRODUCT INHIBITION OF CARBOXYPEPTIDASE A CATALYZED HYDROLYSIS OF O-(TRANS-CINNAMOYL)-L-BETA-PHENYLLACTATE

The pH dependencies of the kinetic constants for the carboxypeptidase A catalyzed hydrolysis of O-(trans-cinnamoyl)-L-β-phenyllactate (IV) were studied over the range pH 5.5-10.2 at 25°. The value of Kcat was found to depend on a base of pKa ~ 6.2 in the enzyme. This compares with the corresponding pKavalue of 7.2 determined earlier for Oacetyl-L-mandelate (III). The error limits on the kcat data obtained in the high pH range were large and made analysis of those results difficult. The value of kcat/Km for IV depends on a base in carboxypeptidase of pKa. ~ 6.5 and on an acid of pKa ~ 9.4. The corresponding dissociation constants for III are ~6.9 and 7.5, respectively. A plot of the pH dependence of the Ki values for the product, L-β-phenyllactate, which is a competitive inhibitor shows an inflection near pH 8.8. The interpretation of these results in terms of the ionization of specific groups at the active site of the enzyme has been considered. © 1969, American Chemical Society. All rights reserved.