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

A time-resolved XAFS investigation has been carried out during substrate hydrolysis of Z-Sar-Phe by carboxypeptidase A (ZnCPD), a zinc containing metalloprotease. Time-dependent XAFS spectral changes observed are consistent with the spectral changes from the freeze-trapped enzyme-substrate intermediate to the free enzyme. The time course of the reaction, 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 difference of cobalt-carboxypeptidase A hydrolysis at the same temperatures. This study demonstrated that XAFS is a valuable tool for characterizing the kinetics of the native ZnCPD catalysis, and that the structure alteration of the intermediates can be directly determined.