Studies on the Active Site of Clostripain

Clostripain is rapidly and selectively inactivated by the chloromethyl ketone derived from cr-N-tosyl-L-lysine (TLCK) with an apparent second order rate constant of 8.7 X lo4 M-I set-i at pH 6.5. Alkylation by TLCK occurs only with catalytically active enzyme, the rate of which is considerably retarded by the competitive inhibitors, oc-Ntosyl-L-homoarginine methyl ester and benzamidine. In contrast, the chloromethyl ketone derived from c+N-tosyl-Lphenylalanine (TPCK) does not inhibit clostripain at molar concentrations 100 times greater than the effective concentration of TLCK. A very large molar excess of TPCK does inhibit the enzyme with an apparent rate constant of 46.7 M-l set-i at pH 6.5. Other irreversible inhibitors of clostripain such as dibromoacetone and iodoacetate require molar concentrations 300 and 3000 times greater, respectively, than TLCK for equivalent inactivation. Such results are in accord with the substrate specificity of the enzyme and support an active site-directed mechanism for inhibition by TLCK. The rapidity of alkylation of the active site of clostripain by TLCK is consistent with the large kcat (643 see-l) observed for substrate hydrolysis (benzoylarginine ethyl ester) and the tentative identification of an essential -SH group in the catalytic mechanism. These results extend the use of TLCK in labeling the active site of a protease with high arginine specificity and offer a method for the specific irreversible inactivation of clostripain which occurs as the major proteolytic contaminant in both crude and highly purified collagenase preparations.