Glucose permease of Escherichia coli. The effect of cysteine to serine mutations on the function, stability, and regulation of transport and phosphorylation.
The glucose permease (IIGlc/IIIGlc complex) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It contains 3 cysteine residues, of which Cys-204 and Cys-326 are localized in the hydrophobic part and Cys-421 in the hydrophilic part of the IIGlc subunit. The cysteines were replaced, one at a time, by serines, and the effect of these mutations on stability, regulation, and catalytic properties of IIGlc was investigated in vivo and in vitro. Cys-204 and Cys-326 are not required for catalytic function and are not involved in the membrane potential-dependent regulation of IIGlc activity (Robillard, G. T., and Konings, W. N. (1982) Eur. J. Biochem. 127, 597-604). Replacement of these cysteines by serines results, however, in reduced stability of IIGlc in vivo (C204S) and in vitro (C204S and C326S), indicating that these substitutions in a hydrophobic environment can destabilize the protein structure. Cys-421 is absolutely required for transport and phosphorylation of glucose. C421S can neither be phosphorylated by phospho-IIIGlc nor catalyze the phosphoryl exchange between [14C] glucose and glucose 6-phosphate at equilibrium. C421S does not interfere with the activity of simultaneously expressed wild-type IIGlc. Unexpectedly C421S and wild-type IIGlc support growth on maltose of Escherichia coli ZSC112L (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199), a strain which otherwise does not grow on this disaccharide as the only carbon source. C421S appears to facilitate the efflux of a growth inhibiting intermediate (glucose?) of maltose. Wild-type IIGlc catalyzes the intracellular phosphorylation of glucose derived from maltose. It is concluded that the cytoplasmic domain of IIGlc interacts with IIIGlc, the cytoplasmic subunit of the glucose permease, and also participates in phosphorylation of glucose, and that phosphorylation occurs independently of transport, although transport of glucose by wild-type IIGlc cannot occur without concomitant phosphorylation.
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D003545:Cysteine; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine
D012694:Serine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D003545:Cysteine