Glucose permease of Escherichia coli. Purification of the IIGlc subunit and functional characterization of its oligomeric forms.
The membrane subunit (IIGlc) of the glucose permease has been purified from overproducing Escherichia coli. About 2 mg of pure protein was obtained from 10 g (wet weight) of cells. IIGlc of E. coli and Salmonella typhimurium are functionally indistinguishable. A small difference was revealed, however, by a monoclonal antibody which neutralizes glucose phosphorylation activity of IIGlc from S. typhimurium, but does not cross-react with IIGlc of E. coli. A dimeric form of purified IIGlc can be detected by chemical cross-linking and by zonal sedimentation at 4 degrees C. Upon mild oxidation a disulfide bond is formed between the subunits of the dimer. Oxidized IIGlc is more stable than the reduced form but is inactive because it cannot be phosphorylated by the cytoplasmic subunit (IIIGlc) of the glucose permease. Cys-421 could be identified as the oxidation-sensitive residue, using a novel assay to detect IIIGlc-dependent phosphorylation of nitrocellulose-bound IIGlc that has been purified by gel electrophoresis. No dimeric form of phosphorylated IIGlc could be detected. Because phosphorylated IIGlc is a catalytic intermediate it is concluded that catalytically active IIGlc is a monomer and that the dimeric form is an artefact observed only with purified resting IIGlc. That IIGlc is active as a monomer is further supported by the observation that monomeric IIGlc catalyzes phosphoryl exchange between glucose and glucose 6-phosphate at equilibrium and that an excess of inactive IIGlc with a serine replacing Cys-421 does not interfere with the activity of wild-type IIGlc as would be expected if interaction between the subunits in a dimer were essential for activity.
D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000302:isolation & purification*; D010766:Phosphorylation; D012486:Salmonella typhimurium / Q000201:enzymology
D046911:Macromolecular Substances; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase