Demonstration of the presence of a histidine residue at the active site of streptococcal proteinase.

Experiments are described which have led to the selective alkylation of a histidine residue in streptococcal proteinase without concomitant reaction with the single essential sulfhydry1 group. The reduced, active form of the enzyme was first treated with sodium tetrathionate to protect the sulfhydry1 group and to introduce a negative charge near the active center. The resulting inactive derivative was not detectably alkylated by iodoacetic acid, iodoacetamide, N-chloroacetyltryptophan, or N-chloroacetylglycyleucine. After treatment with these reagents activity could be regenerated by reduction with a thiol. If, however, the S-sulfenylsulfonate derivative of the enzyme was exposed to a positively charged alkylating agent, or-N-bromoacetylarginine methyl ester, 1 molecule of the reagent was incorporated per molecule of protein, as indicated by an increase in the amount of arginine by 1 residue, a decrease in the amount of histidine by 1 residue, and the formation of 1 residue of 3-carboxymethylhistidine. The modified protein possessed no enzymatic activity when the sulfhydryl group was regenerated by reduction. These experiments thus furnish direct chemical evidence for the involvement of a histidine residue, as well as of a sulfhydryl group, in the active site of streptococcal proteinase.