Soskine Misha, Adam Yoav, Schuldiner Shimon
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
J Biol Chem. 2004 Mar 12;279(11):9951-5. doi: 10.1074/jbc.M312853200. Epub 2003 Dec 29.
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.
本文提出了一种研究底物与离子通量耦合的新方法。EmrE是一种来自大肠杆菌的H(+)耦合多药转运蛋白。去污剂增溶的EmrE以pH依赖模式高亲和力结合底物。在此，我们首次在离子耦合转运蛋白中展示了去污剂增溶制剂中底物诱导的质子释放。直接测量使得对该现象进行重要的定量成为可能。因此，野生型和14位有单个羧基的突变体中质子释放的化学计量非常相似，约为0.8个质子/单体。这些发现表明，参与质子释放的唯一残基是一个高度保守的膜嵌入谷氨酸（Glu-14），并且EmrE功能寡聚体中的所有Glu-14残基都参与质子释放。此外，根据释放的pH依赖性，我们确定Glu-14的pK为8.5，而相同位置天冬氨酸替代物的pK为6.7。14位羧基的高pK对于质子和底物通量的耦合至关重要。
