Frillingos S, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California at Los Angeles 90024-1570, USA.
Biochemistry. 1996 Aug 6;35(31):10166-71. doi: 10.1021/bi960995r.
A carboxylic acid at position 325 in helix X is obligatory for lactose/H+ symport at a step corresponding to deprotonation of lactose permease [Carrasco, N. et al. (1989) Biochemistry 28, 2533-2539]. In this paper, pH profiles for active transport, efflux, and equilibrium exchange are analyzed for wild-type permease and mutant Glu325-->Asp. With respect to active transport and efflux down a concentration gradient, both of which involve net H+ translocation and are defective in the mutant, the wild-type and the mutant exhibit similar profiles, and at no pH is the mutant stimulated relative to the wild-type. Strikingly, exchange which does not involve H+ translocation is comparable in the wild-type and the Glu325-->Asp mutant below pH 7.5. Above pH 7.5, however, the exchange activity of the mutant is progressively and reversibly inhibited with a midpoint at about pH 8.5; while the exchange activity of wild-type permease is only mildly decreased above pH 9.5, and exchange by Glu325-->Ala or Glu325-->Gln permease is comparable to wild-type and unaffected by pH. The findings are consistent with the idea that translocation of the ternary complex between the permease, lactose, and H+ does not tolerate a negative charge at position 325. In wild-type permease, the electrostatic interaction between Glu325 (helix X) and Arg302 (helix IX) is sufficiently strong that the carboxylate is unaffected by pH. In contrast, with Asp at position 325, the electrostatic interaction is broken, the carboxylate becomes protonated, and the acid exhibits a pKa of about 8.5. Monoclonal antibody 4B1 binds to the periplasmic loop between helices VII and VIII of the permease [Sun, J. et al. (1996) Biochemistry 35, 990-998] and mimics the Glu325 mutants. Dramatically, 4B1 shifts the apparent pKa for exchange from about pH 8.5 to 7.5 in the Glu325-->Asp mutant with little or no effect on the wild-type or the Glu325-->Ala mutant. The findings are consistent with the conclusion that the uncoupling effect of 4B1 involves a conformational change in helix VII and/or VIII that secondarily alters the pKa of the essential carboxylic acid at position 325.
螺旋X中325位的羧酸对于乳糖/H⁺同向转运是必需的，该转运过程对应乳糖通透酶的去质子化步骤[卡拉斯科，N.等人（1989年）《生物化学》28卷，2533 - 2539页]。在本文中，分析了野生型通透酶和突变型Glu325→Asp的主动转运、流出及平衡交换的pH曲线。对于主动转运和顺着浓度梯度的流出，这两者都涉及净H⁺转运且在突变体中存在缺陷，野生型和突变型呈现相似的曲线，并且在任何pH下突变体相对于野生型都没有受到刺激。引人注目的是，在pH 7.5以下，不涉及H⁺转运的交换在野生型和Glu325→Asp突变体中是相当的。然而，在pH 7.5以上，突变体的交换活性逐渐且可逆地受到抑制，中点约为pH 8.5；而野生型通透酶的交换活性在pH 9.5以上仅轻微下降，并且Glu325→Ala或Glu325→Gln通透酶的交换与野生型相当且不受pH影响。这些发现与这样的观点一致，即通透酶、乳糖和H⁺之间三元复合物的转运在325位不能容忍负电荷。在野生型通透酶中，Glu325（螺旋X）和Arg302（螺旋IX）之间的静电相互作用足够强，以至于羧酸盐不受pH影响。相比之下，在325位为Asp时，静电相互作用被破坏，羧酸盐质子化，并且该酸表现出约8.5的pKa。单克隆抗体4B1与通透酶螺旋VII和VIII之间的周质环结合[孙，J.等人（1996年）《生物化学》35卷，990 - 998页]并且模拟Glu325突变体。显著的是，4B1将Glu325→Asp突变体中交换的表观pKa从约pH 8.5转变为7.5，而对野生型或Glu325→Ala突变体几乎没有影响。这些发现与以下结论一致，即4B1的解偶联作用涉及螺旋VII和/或VIII的构象变化，进而改变325位必需羧酸的pKa。
