WATER IS REQUIRED FOR PROTON-TRANSFER FROM ASPARTATE-96 TO THE BACTERIORHODOPSIN SCHIFF-BASE
WATER IS REQUIRED FOR PROTON-TRANSFER FROM ASPARTATE-96 TO THE BACTERIORHODOPSIN SCHIFF-BASE
复制标题
DOI：
10.1021/bi00109a023
复制
发表时间：
1991-11-12
期刊：
影响因子：
2.9
通讯作者：
LANYI, JK
中科院分区：
文献类型：
--
作者：
CAO, Y;VARO, G;LANYI, JK
在细菌视紫红质光循环中的MN-> BR反应序列中，质子在D96和Schiff碱之间交换，并且D96从细胞质表面被重质子化。我们探测这些和其他的光循环反应，具有生物活性的溶质和扰动剂，并发现M-N反应是专门抑制从蛋白质中提取水。野生型蛋白质中的N -> BR反应和位点特异性突变体D96 N中席夫碱从细胞质表面的直接再质子化受到的影响要小得多。因此，它似乎是需要水的蛋白质内的反应，其中质子是从一个埋电负性基团分离，但不为那些限速步骤是在蛋白质表面的质子捕获。在野生型中，席夫碱再质子化的最大障碍部分是质子与D96分离的焓，其总计约40 kJ/mol。我们认为，尽管这D96赋予一个整体的动力学优势，因为当这个残基成为阴离子的N状态，其附近的细胞质表面的电场降低了在下一步捕获质子的自由能垒。在D96 N蛋白中，M -> BR反应的屏障比野生型中M -> N和N -> BR部分反应的速率所预期的高20 kJ/mol，大概是因为该机制不可用。
During the M 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 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.