Carboxypeptidase A-Catalyzed Hydrolysis of O-(α-Acylamino-2-Styrylacryloyl)-L-β-Phenyllactates: Search of Specific Ester Substrates Hydrolyzed through Accumulation of Intermediates

Abstract

Results of a previous study on the hydrolysis of O-(α-acylamino-cinnamoyl)-L-β-phenyllactates E1 and E2 catalyzed by carboxypeptidase A (CPA) supported the anhydride mechanism for the action of CPA. Since these esters are hydrolyzed very slowly by CPA, it is desirable to demonstrate that the same mechanism is operative with specific substrates in order to establish the anhydride mechanism firmly. In this study, a search is made for ester substrates that are hydrolyzed very fast by CPA, involve accumulation of anhydride intermediates, and possess good chromophoric properties. O-(α-Acylamino-2-styrylacryloyl)-L-β-phenyllactates H and I, which have been prepared for this purpose, manifested kinetic features such as very small Kappm and pH-independent kcat, which are consistent with the accumulation of anhydride intermediates. When judged with the magnitude of both kcat and kcat/Kappm, H and I can be regarded as the link connecting nonspecific esters E1 and E2 and specific ester G1. The kcat for esters H and I as well as nonspecific esters E1 and E2 and specific ester G1 are independent of pH, and this kinetic common feature may be taken to reflect the rate-determining breakdown of the anhydride intermediate. Results of the present study, therefore, suggest that the anhydride mechanism is also operative in the action of CPA on specific ester substrates.