A triple mutant, K319N/H322Q/E325Q, of the lactose permease cotransports H+ with thiodigalactoside.

In a previous study, we characterized a lactose permease mutant (K319N/E325Q) that can transport H+ ions with sugar. This result was surprising because other studies had suggested that Glu-325 plays an essential role in H+ binding. To determine if the lactose permease contains one or more auxiliary H+ binding sites, we began with the K319N/E325Q strain, which catalyzes a sugar-dependent H+ leak, and isolated third site suppressor mutations that blocked the H+ leak. Three types of suppressors were obtained: H322Y, H322R, and M299I. These mutations blocked the H+ leak and elevated the apparent K m value for lactose. The M299I and H322Y suppressors could still transport H+ with β-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H+/sugar cotransport.