Cao Y, Váró G, Chang M, Ni B F, Needleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717.
Biochemistry. 1991 Nov 12;30(45):10972-9. doi: 10.1021/bi00109a023.
During the M in equilibrium with N----BR reaction sequence in the bacteriorhodopsin photocycle, proton is exchanged between D96 and the Schiff base, and D96 is reprotonated from the cytoplasmic surface. We probed these and the other photocycle reactions with osmotically active solutes and perturbants and found that the M in equilibrium with N reaction is specifically inhibited by withdrawing water from the protein. The N----BR reaction in the wild-type protein and the direct reprotonation of the Schiff base from the cytoplasmic surface in the site-specific mutant D96N are much less affected. Thus, it appears that water is required inside the protein for reactions where a proton is separated from a buried electronegative group, but not for those where the rate-limiting step is the capture of a proton at the protein surface. In the wild type, the largest part of the barrier to Schiff base reprotonation is the enthalpy of separating the proton from D96, which amounts to about 40 kJ/mol. We suggest that in spite of this D96 confers an overall kinetic advantage because when this residue becomes anionic in the N state its electric field near the cytoplasmic surface lowers the free energy barrier of the capture of a proton in the next step. In the D96N protein, the barrier to the M----BR reaction is 20 kJ/mol higher than what would be expected from the rates of the M----N and N----BR partial reactions in the wild type, presumably because this mechanism is not available.
在细菌视紫红质光循环中M与N----BR反应序列处于平衡状态时，质子在D96和席夫碱之间交换，并且D96从细胞质表面重新质子化。我们用具有渗透活性的溶质和扰动剂探究了这些以及其他光循环反应，发现从蛋白质中抽出水会特异性抑制M与N的平衡反应。野生型蛋白质中的N----BR反应以及位点特异性突变体D96N中席夫碱从细胞质表面的直接质子化受影响较小。因此，似乎蛋白质内部对于质子从埋在其中的电负性基团分离的反应是需要水的，但对于限速步骤是在蛋白质表面捕获质子的反应则不需要水。在野生型中，席夫碱重新质子化的最大障碍是将质子从D96分离的焓，其约为40 kJ/mol。我们认为，尽管如此，D96仍赋予了整体动力学优势，因为当该残基在N状态下变为阴离子时，其在细胞质表面附近的电场会降低下一步捕获质子的自由能垒。在D96N蛋白质中，M----BR反应的能垒比根据野生型中M----N和N----BR部分反应速率预期的要高20 kJ/mol，推测是因为这种机制不可用。
