Brown L S, Needleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Biochemistry. 2000 Feb 8;39(5):938-45. doi: 10.1021/bi9921900.
Deuterium kinetic isotope effects (KIE) were measured, and proton inventory plots were constructed, for the rates of reactions in the photocycles of wild-type bacteriorhodopsin and several site-specific mutants. Consistent with earlier reports from many groups, very large KIEs were observed for the third (and largest) rise component for the M state and for the decay of the O state, processes both linked to proton transfers in the extracellular region. The proton inventory plots (ratio of reaction rates in mixtures of H(2)O and D(2)O to that in H(2)O vs mole fraction of D(2)O) were approximately linear for the first and second M rise components and for M decay, as well as for O decay, indicating that the rates of these reactions are limited by simple proton transfer. Uniquely, the third rise component of M (and in the D96N mutant also a fourth rise component) exhibited a strongly curved proton inventory plot, suggesting that its rate, which largely accounts for the rate of deprotonation of the retinal Schiff base, depends on a complex multiproton process. This curvature is observed also in the E194Q, E204Q, and Y57F mutants but not in the R82A mutant. From these findings, and from the locations of bound water in the extracellular region in the crystal structure of the protein [Luecke, Schobert, Richter, Cartailler, and Lanyi (1999) J. Mol. Biol. 291, 899-911], we suspect that the effects of deuterium substitution on the formation of the M state originate from cooperative rearrangements of the extensively hydrogen-bonded water molecules 401, 402, and 406 near Asp-85 and Arg-82.
测定了野生型细菌视紫红质和几个位点特异性突变体光循环中反应速率的氘动力学同位素效应（KIE），并构建了质子丰度图。与许多研究小组早期的报告一致，在M态的第三个（也是最大的）上升组分以及O态的衰减过程中观察到了非常大的KIE，这两个过程都与细胞外区域的质子转移有关。对于M态的第一和第二个上升组分、M态的衰减以及O态的衰减，质子丰度图（H₂O和D₂O混合物中的反应速率与H₂O中的反应速率之比相对于D₂O的摩尔分数）近似呈线性，表明这些反应的速率受简单质子转移的限制。独特的是，M态的第三个上升组分（在D96N突变体中还有第四个上升组分）呈现出强烈弯曲的质子丰度图，这表明其速率（在很大程度上决定了视黄醛席夫碱的去质子化速率）取决于一个复杂的多质子过程。在E194Q、E204Q和Y57F突变体中也观察到了这种曲率，但在R82A突变体中未观察到。基于这些发现，以及蛋白质晶体结构中细胞外区域结合水的位置[Luecke, Schobert, Richter, Cartailler, and Lanyi (1999) J. Mol. Biol. 291, 899 - 911]，我们推测氘取代对M态形成的影响源于靠近Asp - 85和Arg - 82的广泛氢键连接的水分子401、402和406的协同重排。
