KINETIC-STUDY OF CARBOXYPEPTIDASE Y-CATALYZED PEPTIDE CONDENSATION-REACTIONS IN AQUEOUS-ORGANIC SOLVENT

The effects of an organic co-solvent on the carboxypeptidase Y-catalyz ed condensation of Cbz-Phe and Gly-NH2 were investigated with respect to both the apparent equilibrium yield and the kinetics of the condens ation reaction. The highest yield was obtained in a 50% DMSO solution. After measuring the solvent-induced deactivation of the enzyme, the t ime-course of the peptide condensation was examined, and the initial v elocity was analyzed. When the concentration of the carboxyl (C) compo nent was fixed, reciprocal plots showed a linear relationship. Further more, the lines for different concentrations of the fixed components w ere not parallel, th us indicating that the condensation reaction did not proceed by a Ping-Pong Bi-Bi mechanism. With fixed concentrations of the amine (A) component and various concentrations of Cbz-Phe, the initial Velocity did not yield simple saturation profiles, but showed an apparent substrate inhibition. The data for lower concentrations of the C-component indicated that the reciprocal plot intersected in the 3rd quadrant, thus denoting a random Bi-Bi mechanism. These results i ndicate that acyl-enzyme intermediates can be formed from the C-compon ent, either with the free enzyme or with the A-component-bound enzyme, and that aminolysis of the ester bond in the enzyme-C-component inter mediate occurs while water is bound on the enzyme. Therefore, the exis tence of a ternary complex of enzyme/A-component/C-component or enzyme /peptide/water is suggested. The parameters evaluated for such a mecha nism are: cooperative factor (alpha) > 1 (negative cooperativity of bi nding), K-m(c) = ca. 2mM, K-m(A) >> 0.5 M and k(cat)(alpha - 1) = 0.1 5 - 0.2s(-1).