A Peptidase - inactive Derivative of Carboxypeptidase A Modified

The first derivative of carboxypeptidase A, cobalt(III)(ethylenediamine-N,N’-diacetato)(arsanilazotyrosinato 248 (Co(III)(EDDA)(AA-CPA-Zn)), in which only the active site residue tyrosine 248 is blocked, has been shown to be completely peptidase-inactive while retaining esterase activity. Peptides are excellent inhibitors of esterase activity showing Ki values somewhat below the corresponding K,,, values of the native enzyme. Short peptides are noncompetitive inhibitors of both short and long ester hydrolysis by Co(III)(EDDA)(AA-CPA-Zn), implying that the binding positions for esters and peptides are not the same. This behavior is consistent with the differential effects of various inhibitors of peptide and ester hydrolysis in the native enzyme, indicating that substrate binding to Co(III)(EDDA)(AA-CPA-Zn) reflects well the binding in the native enzyme. In contrast, long peptides are competitive inhibitors of both short and long ester hydrolysis. The results are consistent with nonidentical but overlapping binding sites for peptides and esters prior to their respective rate-determining steps. Acetylation of two active site tyrosines in the native enzyme (Simpson, R. T., Riordan, J. F., and Vallee, B. L. (1963) Biochemistry 2,616-622) results in a peptidaseinactive derivative that is distinctly different from Co(III)(EDDA)(AA-CPA-Zn). In contrast to the native and Co(III)EDDA enzymes, peptides and esters appear to occupy a common binding position in the acetylated derivative. Peptides also bind much more poorly to the acetylated enzyme than to the native or Co(III)EDDA enzymes. Similar effects on peptide binding can be brought about by acetylation of Co(III)(EDDA)(AACPA-Zn).