Does the pressure dependence of kinetic isotope effects report usefully on dynamics in enzyme H-transfer reactions?
Does the pressure dependence of kinetic isotope effects report usefully on dynamics in enzyme H-transfer reactions?
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DOI：
10.1111/febs.13193
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发表时间：
2015-08
期刊：
影响因子：
--
通讯作者：
Scrutton NS
中科院分区：
文献类型：
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作者：
Hoeven R;Heyes DJ;Hay S;Scrutton NS
动力学同位素效应（KIE）的温度依赖性已经成为量子隧道酶促H-转移的主要实验探针。在解释中隐含的是H-转移化学与蛋白质环境的动态耦合的假定作用，即所谓的“促进运动/振动假说”。这个想法仍然有争议，其他人质疑促进运动/振动的重要性和/或存在。解决这个问题的新实验方法正在出现，包括使用质量调制酶和时间分辨光谱。KIE的压力依赖性被认为是量子隧穿反应的潜在探针，因为由零点振动能的差异定义的半经典KIE对压力的变化相对不敏感。然而，报告的氢转移反应的压力和温度（p-T）依赖性研究是有限的。在这里，我们扩展并回顾了现有的p-T研究，这些研究利用了定义良好的实验系统，在这些系统中建立了量子力学隧道。这些包括黄素蛋白、醌蛋白、光活化酶和化学模型系统。我们发现，在这些系统中KIE的p-T依赖性之间没有明显的一般趋势。鉴于p-T研究的复杂性，我们得出结论，除了反应速率/KIE的实验测量外，还需要使用确定的（例如X射线）结构进行计算模拟，以指导p-T效应的解释。在提供对H-转移/环境耦合的新见解时，将原子理解与实验速率测量相结合的组合方法将需要在个案基础上进行仔细评估。虽然个别信息，我们得出的结论是，p-T的研究并没有提供更普遍的见解，已经从KIE的温度依赖性的研究。
The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental probe of enzymatic H‐transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H‐transfer chemistry to the protein environment, the so‐called ‘promoting motions/vibrations hypothesis’. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass‐modulated enzymes and time‐resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi‐classical KIEs, which are defined by differences in zero‐point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature (p‐T) dependence studies of H‐transfer reactions are, however, limited. Here, we extend and review the available p‐T studies that have utilized well‐defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light‐activated enzymes and chemical model systems. We show that there is no clear general trend between the p‐T dependencies of the KIEs in these systems. Given the complex nature of p‐T studies, we conclude that computational simulations using determined (e.g. X‐ray) structures are also needed alongside experimental measurements of reaction rates/KIEs to guide the interpretation of p‐T effects. In providing new insight into H‐transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case‐by‐case basis. Although individually informative, we conclude that p‐T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs.