Kinetic Study of Carboxypeptidase Y-Catalyzed Peptide Condensation Reactions in Aqueous-Organic Solvent

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Abstract
The effects of an organic co-solvent on the carboxypeptidase Y-catalyzed condensation of Cbz-Phe and Gly-NH2were investigated with respect to both the apparent equilibrium yield and the kinetics of the condensation reaction. The highest yield was obtained in a 50% DMSO solution. After measuring the solvent-induced deactivation of the enzyme, the time-course of the peptide condensation was examined, and the initial velocity was analyzed. When the concentration of the carboxyl (C) component was fixed, reciprocal plots showed a linear relationship. Furthermore, the lines for different concentrations of the fixed components were not parallel, thus 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 indicate that acyl-enzyme intermediates can be formed from the C-component, either with the free enzyme or with the A-component-bound enzyme, and that aminolysis of the ester bond in the enzyme-C-component intermediate occurs while water is bound on the enzyme. Therefore, the existence of a ternary complex of enzyme/A-component/C-component or enzyme/peptide/water is suggested. The parameters evaluated for such a mechanism are: cooperative factor (α) > 1 (negative cooperativity of binding), Kcm= ca. 2mM, KAm( 0.5 M and kcal(α – 1) == 0.15 - 0.2s-1.
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