Characterization of the double mutant, Val-177/Asn-322, of the lactose permease

The double mutant, Val-177/Asn-322, was investigated with regard to its ability to transport H⁺ and galactosides. In downhill lactose transport assays, the wild-type strain had a K_m value for lactose uptake of 0.9 mM and a V_max of 0.65 μmol lactose/min·mg protein while the mutant had a significantly higher K_m, value of 1.9 mM but a similar V_max of 0.49 μmol/min·mg protein. In spite of its moderate ability to transport lactose downhill, the Val-177/Asn-322 mutant exhibited the striking property of being completely defective in the uphill accumulation of lactose or methyl-β-D-thiogalactopyranoside. Direct measurements of H⁺ transport, however, showed that the mutant's defect in active accumulation is not due to a defect in the ability to transport H⁺ with lactose or methyl-β-D-thiogalactopyranoside. The Val-177/Asn-322 mutant strain had a H⁺:lactose stoichiometry of 0.84 which was similar to that measured in the wild-type strain (0.68). These results are discussed with regard to the role His-322 plays in H⁺ transport, active accumulation of sugars, and sugar recognition.