PMID:14701800
| Citation |
Soskine, M, Adam, Y and Schuldiner, S (2004) Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter. J. Biol. Chem. 279:9951-5 |
|---|---|
| Abstract |
A novel approach to study coupling of substrate and ion fluxes is presented. EmrE is an H(+)-coupled multidrug transporter from Escherichia coli. Detergent-solubilized EmrE binds substrate with high affinity in a pH-dependent mode. Here we show, for the first time in an ion-coupled transporter, substrate-induced release of protons in a detergent-solubilized preparation. The direct measurements allow for an important quantitation of the phenomenon. Thus, stoichiometry of the release in the wild type and a mutant with a single carboxyl at position 14 is very similar and about 0.8 protons/monomer. The findings demonstrate that the only residue involved in proton release is a highly conserved membrane-embedded glutamate (Glu-14) and that all the Glu-14 residues in the EmrE functional oligomer participate in proton release. Furthermore, from the pH dependence of the release we determined the pK of Glu-14 as 8.5 and for an aspartate replacement at the same position as 6.7. The high pK of the carboxyl at position 14 is essential for coupling of fluxes of protons and substrates. |
| Links |
PubMed Online version:10.1074/jbc.M312853200 |
| Keywords |
Antiporters/chemistry; Antiporters/metabolism; Aspartic Acid/chemistry; Biological Transport; Cell Membrane/metabolism; Detergents/pharmacology; Dicyclohexylcarbodiimide/pharmacology; Escherichia coli/metabolism; Escherichia coli Proteins; Glutamic Acid/chemistry; Hydrogen/chemistry; Hydrogen-Ion Concentration; Ions; Kinetics; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Models, Biological; Plasmids/metabolism; Protein Binding; Protons |
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