A TIME-RESOLVED STUDY OF SUBSTRATE HYDROLYSIS OF CARBOXYPEPTIDASE-A

A time-resolved XAFS investigation has been carried out during substra te hydrolysis of Z-Sar-Phe by carboxypeptidase A (ZnCPD), a zinc conta ining metalloprotease. Time-dependent XAFS spectral changes observed a re consistent with the spectral changes from the freeze-trapped enzyme -substrate intermediate to the free enzyme. The time course of the rea ction, characterized by the normalized difference of the two peaks on the absorption edge, can be fitted with a first-order reaction model. The temperature-dependent rates of the reaction differ by 3 times when compared at 288 K and 278 K, which is consistent with the rate differ ence of cobalt-carboxypeptidase A hydrolysis at the same temperatures. This study demonstrated that XAFS is a valuable tool for characterizi ng the kinetics of the native ZnCPD catalysis, and that the structure alteration of the intermediates can be directly determined.