Smirnova Irina, Kasho Vladimir, Sugihara Junichi, Choe Jun-Yong, Kaback H Ronald
Department of Physiology, University of California Los Angeles, Los Angeles, California 90095-7327, USA.
Biochemistry. 2009 Sep 22;48(37):8852-60. doi: 10.1021/bi9011918.
A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K(d)(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K(d)(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH on K(d)(app) inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.
已确定大肠杆菌乳糖通透酶（LacY）与糖结合的亲和力具有约10.5的极高pKa值，这表明在生理条件下，底物与完全质子化的LacY结合。我们现在系统地测试了参与糖结合以及H⁺转运和偶联的残基的定点替换，以确定哪些残基可能导致这种碱性pKa。糖结合位点（Glu126、Trp151、Glu269）中的突变显著降低了对糖的亲和力，但并未改变结合的pKa。相反，参与H⁺转运的残基（Arg302、Tyr236、His322、Asp240、Glu325、Lys319）的替换表现出糖结合的pKa值，这些值要么向中性pH偏移，要么与pH无关。除了Glu325或Lys319的中性替换外，所有突变体的糖结合表观解离常数（K(d)(app)）值都大幅增加，Glu325或Lys319的中性替换的特征是从pH 5.5到pH 11具有极高的糖结合亲和力（即低K(d)(app)）。通过停流荧光法直接测量的糖结合的结合和解离速率常数的pH依赖性表明，k(off)是碱性pH下亲和力变化的主要因素，并证实了从稳态荧光法推断的pH对K(d)(app)的影响。这些结果表明，野生型LacY糖结合的高pKa不能归因于任何单个氨基酸残基，而似乎存在于参与H⁺转运的残基复合物中。有结构证据表明该复合物中结合有水，并且水可能是负责糖结合pH依赖性的质子化位点。
