Departments of Physiology and ‡Microbiology, Immunology & Molecular Genetics, §Molecular Biology Institute, University of California-Los Angeles , Los Angeles, California 90095, United States.
Biochemistry. 2013 Nov 19;52(46):8261-6. doi: 10.1021/bi4013269. Epub 2013 Nov 8.
In this study of the lactose permease of Escherichia coli (LacY), five functionally irreplaceable residues involved specifically in H(+) translocation (Arg302 and Glu325) or in the coupling between protonation and sugar binding (Tyr236, Glu269, and His322) were mutated individually or together with mutant Glu325 → Ala. The wild type and each mutant were purified and reconstituted into proteoliposomes, which were then examined using solid-supported-membrane-based electrophysiology. Mutants Glu325 → Ala or Arg302 → Ala, in which H(+) symport is abolished, exhibit a weakly electrogenic rapid reaction triggered by sugar binding. The reaction is essentially absent in mutant Tyr236 → Phe, Glu269 → Ala, and His322 → Ala, and each of these mutations blocks the electrogenic reaction observed in the Glu325 → Ala mutant. The findings are consistent with the interpretation that the electrogenic reaction induced by sugar binding is due to rearrangement of charged residues in LacY and that this reaction is blocked by mutation of each member of the Tyr236/Glu269/His322 triad. In addition, further support is provided for the conclusion that deprotonation is rate limiting for downhill lactose/H(+) symport.
在这项对大肠杆菌乳糖通透酶（LacY）的研究中，我们分别或共同突变了五个在 H+转运（Arg302 和 Glu325）或质子化和糖结合偶联中起关键作用的、功能上不可替代的残基（Tyr236、Glu269 和 His322）。野生型和每种突变型均被纯化并重新组装到脂质体中，然后使用基于固载膜的电生理学进行检测。突变型 Glu325→Ala 或 Arg302→Ala 中，H+协同转运被完全阻断，其表现出由糖结合引发的、弱电性的快速反应。在突变型 Tyr236→Phe、Glu269→Ala 和 His322→Ala 中，该反应基本不存在，并且这三种突变都能阻断在 Glu325→Ala 突变体中观察到的电反应。这些发现与如下观点一致，即糖结合诱导的电反应归因于 LacY 中带电残基的重排，而该反应被 Tyr236/Glu269/His322 三联体中每个成员的突变所阻断。此外，这进一步支持了去质子化对乳糖/H+协同转运的下坡作用具有限速作用的结论。
