Role of glutamate-269 in the lactose permease of Escherichia coli.
Glu-269, which is located on the hydrophilic face of putative helix VIII in the lactose permease of Escherichia coli, has been replaced with Asp, Gln or Cys by oligonucleotide-directed, site specific mutagenesis. Cells expressing Asp-269 permease exhibit no lactose accumulation or lactose-induced H+ translocation, but retain some ability to mediate lactose influx down a concentration gradient at high substrate concentrations. Furthermore, right-side-out membrane vesicles containing Asp-269 permease do not catalyse active lactose transport, facilitated lactose efflux or equilibrium exchange. Remarkably, however, Asp-269 permease accumulates beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside in a partially uncoupled fashion, whereas no transport of methyl-beta,D-thiogalactopyranoside, sucrose or maltose is detectable. Mutant permeases containing neutral replacements (Gln or Cys) or Glu-269 are completely devoid of activity, although the proteins are present in the membrane at concentrations comparable with wild-type or Asp-269 permease. The observations demonstrate that a carboxylate at position 269 is essential for transport activity, and Glu-269 is important for substrate binding and/or recognition.
Ujwal
M L
ML
Howard Hughes Medical Institute, Department of Physiology, University of California at Los Angeles 90024.
Sahin-Tóth
M
M
Persson
B
B
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D017931:DNA Primers / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005971:Glutamates / Q000737:chemistry / Q000235:genetics; D018698:Glutamic Acid; D017136:Ion Transport; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D027981:Symporters
D017931:DNA Primers; D004187:Disaccharides; D029968:Escherichia coli Proteins; D005971:Glutamates; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D018698:Glutamic Acid; C021499:lactose permease; D007785:Lactose