Vicinal dithiol-disulfide distribution in the Escherichia coli mannitol specific carrier enzyme IImtl

Abstract

E. coli mannitol specific carrier enzyme II (EIImtl) in membrane vesicles can be inhibited by the action of the oxidizable substrate-reduced phenazine methosulfate (PMS) in a manner similar to E. coli glucose specific enzyme II. The fact that reduced PMS and various oxidizing agents protect the enzyme from inactivation by the sulfhydryl reagents N-ethylmaleimide and bromopyruvate suggests that the active form possesses a dithiol which can be protected by conversion to a disulfide. The SH-disulfide distribution was examined in purified EIImtl by labeling studies with N-[1-14C]ethylmaleimide ([14C]NEM). EIImtl can be alkylated at 3 positions per peptide chain. When alkylation takes place in 8 M urea, only 2 positions are labeled. The 3rd position becomes labeled in urea only after treatment with dithiothreitol, suggesting that the native enzyme is composed of 2 subunits linked by a disulfide bridge. The remaining 2 SH groups per peptide chain appear to undergo changes in oxidation state. Treatment of the active enzyme with NEM leads to complete inactivation and incorporation of 1 mol of [14C]NEM per peptide chain. Oxidizing agents protect the activity and prevent labeling presumably by forming a disulfide. Phosphorylating the enzyme (1 phosphoryl group per peptide chain) fully protects the activity, but 1 mol of NEM per peptide chain is still incorporated. Subsequent dephosphorylation by adding mannitol causes a 2nd mol of [14C]NEM to be incorporated and results in complete inactivation. The labeled site in the phosphorylated enzyme can also be protected by oxidizing agents. The possibility that dithiol-disulfide interchange occurs during the turnover of the carrier is discussed.