Evidence for the Existence of a Channel in the Glucose-Specific Carrier EIIGlc of the Salmonella typhimurium Phosphoenolpyruvate-Dependent Phosphotransferase System

The effect of membrane-impermeable sulfhydryl reagents on glucose-specific enzyme II (EIIGlc) activity has been studied in Salmonella typhimurium whole cells and in properly sealed inverted cytoplasmic membrane vesicles. Glutathione N-hexylmaleimide and N-polymethylenecarboxymaleimides inactivate methyl α-D-glucopyranoside (α-MeGlc) transport and phosphorylation in whole cell preparations at a dithiol that can be protected by oxidizing reagents, trivalent arsenicals, or phosphorylation of EIIGlc. Accessibility to this activity-linked site is restricted to small apolar reagents or to polar reagents with a hydrophobic spacer between the polar group and the reactive maleimide moiety. These same reagents inactivate α-MeGlc phosphorylation in inverted cytoplasmic membrane vesicles. Inhibition results from reaction at a dithiol that can be protected by nonpermeant mercurials, oxidants, and arsenicals as well as by phosphorylation of EII. The characteristics of this site are virtually identical with those of the activity-linked dithiol elucidated in intact cells. No evidence could be found for a second activity-linked site on the other side of the membrane when the permeable reagent N-ethylmaleimide was used. Since only one activity-linked dithiol can be detected with sealed inverted membrane vesicles or intact cells and it is accessible to membrane-impermeable sulfhydryl reagents from both sides of the cytoplasmic membrane, we suggest that it is located in a channel constructured by the carrier and that the channel spans the membrane. A second dithiol, not essential for activity, is located near the outer surface of the cytoplasmic membrane

Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System. Evidence That the Dimer Is the Active Form of Enzyme I

In vitro kinetic measurements have been performed by using purified HPr, EI, and a membrane fraction of EII from the Escherichia coli phosphoenolpyruvate-dependent sugar transport system. These measurements reveal very large lag times in the formation of methyl α-glucoside phosphate which are a function of the EI and the EII concentrations. The lag times decrease with increasing concentrations of EI but they increase with increasing concentrations of EII. When EI, together with Mg2+ and phosphoenolpyruvate, is preincubated at 37 °C before starting the kinetic measurements, the lag time can be decreased or eliminated. We have shown that the process responsible for the lag time is the activation of EI by dimerization which is influenced by Mg2+ and phosphoenolpyruvate