Temperature Dependence of Hydride Kinetic Isotope Effects in Solution to Test the Proposed Role of Protein Dynamics in Enzyme Catalysis

项目总结 最近提出的蛋白质动力学与酶反应的化学相结合，提出了一种新的 酶促速度加快的可能来源。在催化中发现这样的物理作用，如果有的话，是 对酶催化理论发展的重要性，这些理论可以指导未来设计高效的 毒品和生物催化剂。研究起源的一种策略是使用酶催化的氢隧道反应，这些反应是 对供体-受体距离(DADS)敏感，从而对任何可以对DADS进行H-DNA采样的蛋白质运动敏感 隧道将会发生。在当代的氢隧穿理论中，较重的氢同位素的隧穿需要 较短的DAD，导致同位素速率差异，从而产生动力学同位素效应(KIE)。因此，Kie是一个 爸爸的作用。因此，对Kie的温度(T)依赖性的研究可以用来反映如何 酶的动态会影响DAD的分布，从而影响酶的化学组成。超过了 在过去的二十年里，人们经常发现Kies是T不依赖的，具有各种野生型酶 但对于不同的变种，变得不同程度地依赖于T。在这些理论中，T-独立的Kies 根据DADS的狭窄分布解释了由于强烈的酶活性部位压缩 效应，而变异中强烈的T依赖KIE对应于广泛分布的DADS 来自自然的动力效应的(部分)损失。当支持这些解释的证据出现时 然而，随着这些KIE工具的积累，使用这些工具来评估催化的物理起源一直是热门的 辩论过了。用其他氢转移/隧穿理论对结果的模拟给出了另一种解释。我们 认为关于Kie的T依赖性与酶中DAD采样的相关性的想法是可以检验的 通过研究DADS可由结构和溶剂控制的溶液中的“较简单”反应 效果。我们的长期目标是设计溶液中的氢转移反应，以复制T依赖的 Kie与变异体的Kie比较，以找出Kie的观察是由还是部分由 提出了酶的偶联动力学。假设是一个更严格的氢转移系统，而不是更广泛 人口众多的父亲导致对Kies的T依赖程度较低。具体的目标是使用电子的，立体的， 溶剂和远程重基团振动效应以逐步调节系统刚性来研究 假设。将选择NADH/NAD辅酶类似物的氢化物转移反应进行研究，以便 其结果可以更直接地与酶的结果进行比较。将确定反应的动力学。 从光谱上看。结果将为关于是否存在酶活性部位的争论提供洞察力 压缩效果。该项目的另一个意义在于，前所未有的对 KIES的结构/溶剂与T依赖性的关系将开辟一个新的研究方向 帮助找到合适的模型来描述溶液和酶中的氢化物隧道化学。

PROJECT SUMMARY Recently proposed protein dynamics coupled to the chemistry of the enzymatic reactions suggests a new possible origin for the enzymatic rate accelerations. Finding such a physical role in catalysis, if any, is of importance to the development of theories for enzyme catalysis that can guide future efforts at design of efficient drugs and biocatalysts. One strategy to study the origin uses enzyme catalyzed H-tunneling reactions that are sensitive to donor-acceptor distances (DADs) and thus to any protein motions that can sample the DADs for H- tunneling to occur. Within the contemporary H-tunneling theories, tunneling of a heavier H isotope requires a shorter DAD, which results in an isotopic rate difference thus a kinetic isotope effect (KIE). As a result, KIE is a function of DAD. Therefore, study of the temperature (T) dependence of KIEs could be used to reflect how enzyme dynamics affect the DAD distributions and thus whether they affect the chemistry of enzymes. Over the past two decades, it has been frequently found that KIEs are T-independent with a variety of wild-type enzymes but become T-dependent to different degrees for different variants. Within those theories, T-independent KIEs have been explained in terms of the narrowly distributed DADs due to a strong enzyme active site compression effect, whereas the strongly T-dependent KIEs in variants correspond to the broadly distributed DADs resulted from the (partial) loss of the dynamical effects from nature. While evidences to support the explanations appear being piled up, use of such KIE tools to evaluate this physical origin for catalysis has, however, been hotly debated. Simulations of the results with other H-transfer/tunneling theories suggest alternative explanations. We regard that ideas about the correlations of T-dependence of KIEs with DAD sampling in enzymes could be tested by study of the “simpler” reactions in solution, for which DADs could be controlled by structural and solvent effects. Our long-term objective is to design H-transfer reactions in solution to replicate the T-dependence of KIEs in enzymes versus variants so as to find whether the KIE observations are caused, or partly caused, by the proposed enzyme’s coupled dynamics. The hypothesis is that a more rigid H-transfer system with less broadly populated DADs gives rise to a weaker T-dependence of KIEs. The specific aims are to use electronic, steric, solvent and remote heavy group vibrational effects to progressively mediate system rigidities to investigate the hypothesis. Hydride transfer reactions of NADH/NAD+ coenzyme analogues will be chosen for the study so that the results can be more directly compared with those from enzymes. Kinetics of the reactions will be determined spectroscopically. Results will provide insight into the argument about whether there is an enzyme active site compression effect. The other significance of the project is that the unprecedented systematic study of the relationship between structure/solvent and T-dependence of KIEs will open a new research direction that could help find appropriate models to describe the hydride tunneling chemistry in both solution and enzymes.