Streptococcal proteinase-catalyzed hydrolysis of some ester and amide substrates.

Streptococcal proteinase was shown to belong to a group of enzymes such as trypsin, chymotrypsin, and subtilisin that exhibit a high ratio of esterase to peptidase activity. In this respect, streptococcal proteinase is different from other sulfhydryl enzymes, such as papain and ficin, and also different from pepsin. These enzymes hydrolyze a specific ester and amide substrate at comparable rates. A spectrophotometric method is described for following the streptococcal proteinase-catalyzed hydrolysis of substrates of the general type benzyloxycarbonyl-X-Y, where x is lysine, norleucine, and glutamic acid, and Y is phenyl, pnitrophenyl, aniline, and p-nitroaniline. This method was used to demonstrate that streptococcal proteinase is an efficient esterase in the cleavage of phenyl and p-nitrophenyl esters, and that in the cleavage of these esters the enzyme is favored by the presence of a cationic group in X. Phenyl esters were found to be more versatile substrates than pnitrophenyl esters for the proteinase, since autolysis of phenyl ester substrates was not observed even at higher pH values (pH 9.0). The effect of pH on the proteinase-catalyzed hydrolyses of benzyloxycarbonylysyl -p nitrophenyl esters was examined. The data revealed that the ratio of log kat/K,,, for the enzyme depends on an ionizable group with a pK of 4.8. When the leaving group, Y, in A-X-Y is changed from phenyl to p-nitrophenyl, kcat/K,,, values increase S-fold. However, when Y is changed from aniline to p-nitroanlline, the kcat/Km value unexpectedly decreases 36-fold. Wang’s theory of pretransition state protonation was used to interpret these data, and it is proposed that streptococcal proteinase hydrolyzes specific ester or amide substrates by different mechanisms. Tosylnorleucylphenylalanine and tosyllysylphenyl ester were found to be resistant to cleavage by streptococcal proteinase. These two derivatives are competitive inhibitors of streptococcal proteinase, with KI values near the kinetically determined value of Kmcapp) for the corresponding benzyloxycarbonyl derivatives.