Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter
Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter
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DOI：
10.1074/jbc.m312853200
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发表时间：
2004-03-12
影响因子：
4.8
通讯作者：
Schuldiner, S
中科院分区：
文献类型：
--
作者：
Soskine, M;Adam, Y;Schuldiner, S
提出了一种新的研究衬底流与离子流耦合的方法。EmrE是来自大肠杆菌的H+偶联多药物转运蛋白。洗涤剂增溶的EmrE以pH依赖性模式以高亲和力结合底物。在这里，我们表明，第一次在离子耦合转运，底物诱导释放的质子在洗涤剂溶解的准备。直接测量允许对该现象进行重要的定量。因此，在野生型和在位置14处具有单个羧基的突变体中的释放的化学计量非常相似，约为0.8个质子/单体。研究结果表明，参与质子释放的唯一残基是高度保守的膜嵌入的谷氨酸（Glu-14），并且EmrE功能性寡聚体中的所有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，天冬氨酸置换在相同位置的pK为6.7。14位羧基的高pK对于质子和底物通量的耦合是必不可少的。
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.