Smirnova Irina N, Kasho Vladimir, Kaback H Ronald
Department of Physiology and Microbiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-7327, USA.
Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):8896-901. doi: 10.1073/pnas.0803577105. Epub 2008 Jun 20.
The effect of bulk-phase pH on the apparent affinity (K(d)(app)) of purified wild-type lactose permease (LacY) for sugars was studied. K(d)(app) values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K(d)(app) for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pK(a) of approximately 10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H(+) symport in either direction across the membrane.
研究了本体相pH对纯化的野生型乳糖通透酶（LacY）与糖类的表观亲和力（K(d)(app)）的影响。K(d)(app)值通过位于远离糖结合位点的两种共价结合荧光报告分子之一的荧光配体诱导变化来确定。在5.5至11的pH范围内测定了三种不同吡喃半乳糖苷的K(d)(app)。所有糖类均获得了约10.5的极高pK(a)。从pH 6至10测量的硫代二半乳糖苷结合的动力学数据表明，在碱性pH下对糖的亲和力降低具体是由于逆向速率增加。通过直接结合测定法对硝基苯基半乳糖苷也观察到类似的效果。由于在pH 5.5至pH 9.0范围内对糖的亲和力保持恒定，因此可以得出结论，在生理pH条件下，就糖结合而言，LacY是完全质子化的。这些结果与以下结论一致，即在乳糖/H(+)双向跨膜共转运过程中，LacY在糖结合之前就已质子化。
