The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase
The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase
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DOI：
10.1098/rstb.2006.1871
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发表时间：
2006-08-29
影响因子：
6.3
通讯作者：
Kohen, Amnon
中科院分区：
文献类型：
--
作者：
Wang, Lin;Goodey, Nina M.;Kohen, Amnon
大肠杆菌二氢叶酸还原酶（ecDHFR）的残基M42和G121位于催化中心的相对侧（分别距催化中心15和19埃）。理论研究表明，这些远端残基可能是一个动力学网络的一部分，耦合到在活性位点催化的反应。ecDHFR突变体G121 V已被广泛研究，似乎对速率有显著影响，但对H-转移的性质只有轻微影响。本工作探讨了M42 W对催化氢化物转移步骤的物理性质的影响。本征动力学同位素效应（KIEs），它们的温度依赖性和激活参数进行了研究。这里提出的研究结果是根据环境耦合的氢隧道效应。与野生型（WT）相比，需要供体-受体距离的波动，导致KIE和放气拦截的显着温度依赖性。M42 W和G121 V与WT酶的比较显示，相对于WT酶，降低的速率、膨胀的初级KIE和它们的温度依赖性是由不完美的电势表面预排列引起的。显然，酶的动力学与反应坐标的耦合被突变改变，支持整个蛋白质的动力学与其催化的化学反应耦合的模型。
Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 angstrom away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor-acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121 V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface prearrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.