Vicinal dithiol-disulfide distribution in the Escherichia coli mannitol specific carrier enzyme IImtl.
Escherichia coli mannitol specific EII in membrane vesicles can be inhibited by the action of the oxidizable substrate-reduced phenazine methosulfate (PMS) in a manner similar to E. coli enzyme IIGlc [Robillard, G. T., & Konings, W. (1981) Biochemistry 20, 5025-5032]. 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 sulfhydryl-disulfide distribution has been examined in purified EIImtl by labeling studies with N-[1-14C]ethylmaleimide ( [14C]NEM). EIImtl can be alkylated at three positions per peptide chain. When alkylation takes place in 8 M urea, only two positions are labeled. The third position becomes labeled in urea only after treatment with DTT, suggesting that the native enzyme is composed of two subunits linked by a disulfide bridge. The remaining two sulfhydryl groups per peptide chain appear to undergo changes in oxidation state as indicated by the following results. (1) 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. (2) Phosphorylating the enzyme (one 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 second mole of [14C]NEM to be incorporated and results in complete inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
D002250:Carbon Radioisotopes; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000378:metabolism; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D002250:Carbon Radioisotopes; D004220:Disulfides; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D005033:Ethylmaleimide; D004229:Dithiothreitol