Adam Yoav, Tayer Naama, Rotem Dvir, Schreiber Gideon, Schuldiner Shimon
Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):17989-94. doi: 10.1073/pnas.0704425104. Epub 2007 Nov 2.
EmrE is an Escherichia coli H(+)-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP(+), a high-affinity substrate, is 2 x 10(7) M(-1).s(-1), a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the k(off) of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.
EmrE是一种大肠杆菌H⁺偶联多药转运蛋白，由于其体积小、稳定性好，且其活性可在去污剂溶液中进行研究，因此提供了一个独特的实验范例。在这项工作中，我们报告了对EmrE中底物结合和底物诱导质子释放的瞬态动力学的研究。为此，我们测量了底物结合时色氨酸荧光的瞬态变化以及底物诱导质子释放的速率。第63位必需且完全保守的色氨酸残基的荧光对质子或底物与结合位点的占据情况敏感。高亲和力底物TPP⁺与去污剂溶解的EmrE的最大结合速率为2×10⁷ M⁻¹·s⁻¹，这是扩散限制反应的典型速率。对中低亲和力底物的速率测量表明，亲和力主要由底物的解离常数（k(off)）决定。在碱性pH下，底物结合速率和底物诱导的质子释放速率较快，而在较低pH下较慢。这些发现表明，底物结合速率由能够结合的物种的生成决定；这是由第14位谷氨酸对质子的高亲和力控制的，因为在相同pH下，用较低pK的天冬氨酸替代时反应更快。
