PMID:11420608
| Citation |
Johnson, JL, Lockheart, MS and Brooker, RJ (2001) A triple mutant, K319N/H322Q/E325Q, of the lactose permease cotransports H+ with thiodigalactoside. J. Membr. Biol. 181:215-24 |
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| Abstract |
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 Km value for lactose. The M299I and H322Y suppressors could still transport H+ with beta-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H+/sugar cotransport. Four mutant strains containing a nonionizable substitution at codon 322 (H322Q) were analyzed. None of these were able to catalyze uphill accumulation of lactose, however, all showed some level of substrate-induced proton accumulation. The level seemed to vary based on the substrate being analyzed (lactose or TDG). Most interestingly, a triple mutant, K319N/H322Q/E325Q, catalyzed robust H+ transport with TDG. These novel results suggest an alternative mechanism of lactose permease cation binding and transport, possibly involving hydronium ion (H3O+). |
| Links | |
| Keywords |
Amino Acid Substitution/genetics; Cations, Monovalent/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Glutamine/genetics; Glutamine/metabolism; Hydrogen/metabolism; Hydrogen-Ion Concentration; Ion Transport; Kinetics; Lactose/metabolism; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed/genetics; Phenotype; Point Mutation/genetics; Protons; Suppression, Genetic/genetics; Symporters; Thiogalactosides/metabolism |
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