Role of glutamate-269 in the lactose permease of Escherichia coli

Abstract

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 β,d-galactopyranosyl 1-thio-β,d-galactopyranoside in a partially uncoupled fashion, whereas no transport of methyl-β,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.

Key Words:

• Active transport
• symport
• site-directed mutagenesis