Evidence for the participation of a sulfhydryl group in the beta-methylaspartase reaction.

Abstract

Summary 1. The participation of an enzyme sulfhydryl group in the reaction catalyzed by β-methylaspartase ( L -threo-3-methylaspartate ammonia-lyase, EC 4,3.1.2) has been investigated by the photooxidation technique and by alkylation studies with N-ethylmaleimide. 2. Photooxidation of the enzyme in the presence of methylene blue yielded a rate constant for enzyme activity loss which was nearly identical with the rate constant for enzyme sulfhydryl destruction. 3. The addition of substrate to the photooxidation system greatly retarded enzyme destruction, indicating that the oxidation-sensitive group(s) lie sufficiently near the active site to be protected by substrate. 4. The enzyme was inactivated by the −SH alkylating agent, N-ethylmaleimide; inactivation was prevented by preincubating the enzyme with certain of its substrates or pseudo-substrates. 5. Effective protectors of −SH have structural similarities around the β (C-3) carbon atom which suggests that protection of -SH involves shielding of an enzyme functional group which lies near the β carbon atom of the substrate in the enzyme-substrate complex.