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

项目摘要 最近提出的蛋白质动力学与酶促反应的化学反应相结合，提出了一种新的 酶促速率加速的可能来源。找到这样一个物理作用的催化，如果有的话，是 重要的是，酶催化理论的发展，可以指导未来的努力，在设计有效的 药物和生物催化剂。研究起源的一种策略是使用酶催化的H-隧道反应， 对供体-受体距离（DAD）敏感，因此对可以对DAD进行H-采样的任何蛋白质运动敏感。 隧道发生。在当代的H隧穿理论中，较重的H同位素的隧穿需要一个 更短的DAD，这导致同位素速率差异，从而导致动力学同位素效应（KIE）。因此，KIE是一个 DAD的功能。因此，KIE的温度（T）依赖性的研究可以用来反映如何 酶动力学影响DAD分布，从而影响它们是否影响酶的化学性质。来 在过去的二十年里，人们经常发现KIE与各种野生型酶都是T无关的 但对于不同的变体，在不同程度上依赖于T。在这些理论中，T独立KIE 已经解释了由于强烈的酶活性位点压缩而导致的狭窄分布的DAD 效应，而变体中的强T依赖性KIE对应于所产生的广泛分布的DAD 从自然界的动力学效应的（部分）损失。虽然支持解释的证据出现了， 然而，利用这些KIE工具来评估催化作用的物理起源， 辩论。使用其他氢转移/隧道理论对结果进行模拟，提出了替代解释。我们 我认为，关于KIE的T依赖性与酶中DAD采样的相关性的想法可以得到检验 通过研究溶液中的“简单”反应，可以通过结构和溶剂控制DAD 方面的影响.我们的长期目标是设计溶液中的H-转移反应，以复制 酶与变体中的KIE，以确定KIE观察结果是否由酶引起或部分由酶引起。 提出了酶的耦合动力学。这一假设是，一个更严格的H-转移系统， 填充的DAD引起KIE的较弱的T依赖性。具体目标是利用电子，空间， 溶剂和远程重基振动效应，以逐步介导系统刚性， 假说.选择NADH/NAD+辅酶类似物的氢化物转移反应进行研究， 结果可以更直接地与来自酶的结果进行比较。将测定反应的动力学 光谱学上结果将提供深入了解的争论是否有一个酶的活性位点 压缩效应该项目的另一个意义是， KIEs的结构/溶剂与温度依赖性之间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穿化学。

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.