A Triple Mutant, K319N/H322Q/E325Q, of the Lactose Permease Cotransports H+ with Thiodigalactoside
| Autor: | Robert J. Brooker, Jerry L. Johnson, M.S.K. Lockheart |
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| Rok vydání: | 2001 |
| Předmět: |
Lactose permease
Cation binding Monosaccharide Transport Proteins Physiology Glutamine Mutant Biophysics Lactose Thiogalactosides chemistry.chemical_compound Suppression Genetic Escherichia coli Point Mutation Sugar transporter Binding site Ion transporter Ion Transport Symporters Escherichia coli Proteins Membrane Transport Proteins Cell Biology Cations Monovalent Hydrogen-Ion Concentration Kinetics Phenotype Amino Acid Substitution chemistry Biochemistry Mutagenesis Site-Directed Protons Cotransporter Hydrogen |
| Zdroj: | The Journal of Membrane Biology. 181:215-224 |
| ISSN: | 0022-2631 |
| DOI: | 10.1007/s00232-001-0024-2 |
| Popis: | 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. 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+). |
| Databáze: | OpenAIRE |
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