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

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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.

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