Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli

Acidic residues which are found on transmembrane segments within the lactose permease may play an important role in H⁺ and/or sugar recognition. To examine the functional roles of Glu-269 and Glu-325, we have constructed a variety of amino acid substitutions (e.g. aspartate, glycine, alanine, serine, or glutamine) via site-directed mutagenesis. At position 269, all mutations appear to have a detrimental effect on sugar affinity, downhill transport, and counterflow. The Asp-269 mutant was able to accumulate lactose against a concentration gradient, whereas all of the nonionizable substitutions at position 269 were completely defective. Nevertheless, in spite of their inability to actively accumulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H⁺ upon the addition of galactosides. Mutations at position 325 had a markedly different phenotype. For example, the Asp-325, Gly-325, and Gln-325 mutants exhibited an apparent K(m) for lactose transport (e.g. 0.21, 0.47, and 0.50 mM, respectively), which was actually lower than that of the wild-type strain (1.44 mM). In counterflow assays, all position 325 mutants also appear to catalyze lactose exchange. Similar to the results obtained at position 269, the Asp-325 mutant exhibited moderate levels of accumulation, whereas none of the nonionizable mutations at position 325 were able to accumulate galactosides against a concentration gradient. However, unlike the position 269 mutants, no H⁺ transport was observed in the Gly-325, Ala-325, Ser-325, or Gln-325 strains upon the addition of lactose, S-β-D-galactopyranosyl-(1,1)-β- thiogalactopyranoside. 1-O-methyl-β-D-galactopyranoside, or melibiose. Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to ΔpH. Overall, these results are consistent with the notion that an acidic residue at position 325 is required for H⁺ transport via the lactose permease. Alternative hypotheses are also discussed.