ROLE OF GLUTAMATE-269 IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Glu-269, which is located on the hydrophilic face of putative helix VI II in the lactose permease of Escherichia coli, has been replaced with Asp, Gln or Cys by oligonucleotide-directed, site specific mutagenesi s. Cells expressing Asp-269 permease exhibit no lactose accumulation o r lactose induced H+ translocation, but retain some ability to mediate lactose influx down a concentration gradient at high substrate concen trations. Furthermore, right-side-out membrane vesicles containing Asp -269 permease do not catalyse active lactose transport, facilitated la ctose efflux or equilibrium exchange. Remarkably, however, Asp-269 per mease accumulates beta,D-galactopyranosly 1-thio-beta,D-galactopyranos ide in a partially uncoupled fashion, whereas no transport of methyl-b eta,D-thiogalactopyranoside, sucrose or maltose is detectable. Mutant permeases containing neutral replacements (Gin or Cys) or Glu-269 are completely devoid of activity, although the proteins are present in th e membrane at concentrations comparable with wild-type or Asp-269 perm ease. The observations demonstrate that a carboxylate at position 269 is essential for transport activity, and Glu-269 is important for subs trate binding and/or recognition.