Evidence for a Carboxyl Group at the Active Center of Bovine Carboxypeptidase B

Bovine carboxypeptidase B has been alkylated at the active center by α-N-bromoacetyl-d-arginine-5-14C. The enzyme was treated at pH 7.0 with a 40-fold molar excess of the reagent. Subsequent fractionation of the reaction mixture on Sephadex G-25 and l-leucyl-d-arginine-Sepharose 4B columns has shown that inactivation is accompanied by incorporation of 1.09 moles of alkylating agent per mole of enzyme. Amino acid analyses of acid and basic hydrolysates of inactivated protein were identical with those of the native enzyme except for the presence of 1 extra residue of arginine. No amino acid was present in lower amounts nor were any new ninhydrin-positive peaks observed. Treatment with 2 m hydroxylamine at pH 9.0 partially reactivated the alkylated enzyme with the concomitant release of 1 equivalent of a 14C-labeled compound and the incorporation of 1 equivalent of hydroxamic acid. The radioactive compound was hydrolyzed in acid to release glycolic acid and arginine at a ratio of 0.94:1.00 and co-chromatographed with synthetically prepared α-N-glycolyl-d-arginine-5-14C on Dowex 50-X4. These results suggest that a carboxyl group of carboxypeptidase B is alkylated by α-N-bromoacetyl-d-arginine-5-14C.