Evidence for an active-center cysteine in the SH-proteinase alpha-clostripain through use of N-tosyl-L-lysine chloromethyl ketone.

The rapid reaction of alpha-clostripain with tosyl-L-lysine chloromethyl ketone results in a complete loss of activity and in the disappearance of one titratable SH group whereas the number of histidine residues is not affected. Tosyl-L-phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride have no effect on the catalytic activity. From the molar ratio and under the assumption of 1:1 molar interaction, the fully active enzyme has a specific activity of 650-700 units/mg [twice the value proposed by Porter et al. (J. Biol. Chem. 246 (1971) 7675-7682)]. Partial oxidation makes it experimentally impossible to attain this maximal value.