Properties of the Single Sulfhydryl Group of Streptococcal Proteinase

In order to investigate the nucleophilic reactivity of the single sulfhydryl group of streptococcal proteinase, the rates of alkylation of this group by chloroacetic acid and chloroacetamide have been studied. The pH dependence of rate constants for alkylation by these two reagents, one anionic and one neutral, were compared with each other and with the pH dependence of rate constants for alkylation of the sulfhydryl of reduced glutathione by the same reagents. The pK of the sulfhydryl group of the enzyme is almost certainly higher than 8.0. The protein sulfhydryl group is 50 to 100 times as reactive as the sulfhydryl in glutathione. The curve of k2 with respect to pH is sigmoid when the protein is alkylated by chloroacetamide and bell-shaped when chloroacetic acid is the alkylating agent. Both reactions lead solely to the formation of the corresponding S-substituted cysteine derivative. The rate curves with respect to pH for the alkylation of reduced glutathione by the two reagents do not show such disparities. The data suggest that the immediate environment of the unusually reactive sulfhydryl group of the enzyme changes from a positive one at pH 4 to a negative one at pH 10. These results show that the ionic environment of the sulfhydryl group in the protein is very complex.