Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase
Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase
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DOI：
10.1073/pnas.0606976103
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发表时间：
2006-10-24
影响因子：
11.1
通讯作者：
Kohen, Amnon
中科院分区：
文献类型：
--
作者：
Wang, Lin;Goodey, Nina M.;Kohen, Amnon
现代酶学中最有趣的问题之一是酶动力学是否进化以增强催化的化学转化。在这项研究中，二氢叶酸还原酶，一种催化单个C-H-C转移的小单体蛋白，被用作模型系统来解决这个问题。实验和计算研究提出了一个动态网络，包括两个远离活性位点的残基（G121和M42）。本研究比较了WT酶的h转移步骤的性质，两个单突变体和它们的双突变体。通过测定本征动力学同位素效应、它们的温度依赖性和激活参数，研究了量子力学隧道效应和酶动力学对h转移步骤的贡献。结果表明，这四种酶具有不同的环境耦合隧穿模式。研究结果表明，自然进化的WT二氢叶酸还原酶不需要供体-受体距离波动（没有门控）。这两种单突变都会在隧穿之前影响系统的重排，因此需要一些门控，但环境耦合隧穿的总体性质与WT酶相似。另一方面，双突变似乎引起H转移性质的重大变化，导致重组不良和大量的门控。这些发现支持了这些远端残基协同影响酶活性位点H转移的建议。这一观察结果与这样一种观点是一致的，即这些远程残基是与催化化学相耦合的动态网络的一部分。
One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes a single C-H-C transfer, is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor-acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry.