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.