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

溶液中氢化物动力学同位素效应的温度依赖性，以测试蛋白质动力学在酶催化中的拟议作用

• 批准号: 10580264
• 负责人: Yun Lu
• 金额: $ 43.35万
• 依托单位: SOUTHERN ILLINOIS UNIV AT EDWARDSVILLE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580264
• 关键词: Acceleration; Active Sites; Affect; Area; Binding; Biochemical Reaction; Catalysis; Charge; Chemicals; Chemistry; Coenzymes; Complex; Coupled; Data; Dependence; Development; Enzymes; Frequencies; Future; Goals; Hydrogen Bonding; Hydroxyl Radical; Isotopes; Kinetics; Link; Literature; Mediating; Methodology; Modeling; Molecular Conformation; Motion; Mutate; NADH; Nature; Pharmaceutical Preparations; Protein Dynamics; Proteins; Reaction; Research; Role; Sampling; Solvents; Structure; System; Temperature; Testing; Theoretical model; Variant; alkyl group; base; charge transfer complex; design; enzyme model; flexibility; insight; light effects; simulation; theories; tool; vibration

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.

项目摘要 最近提出的蛋白质动力学与酶促反应的化学耦合表明一种新的 酶促加速度的可能起源。在催化中找到这种物理作用（如果有的话） 对于开发酶催化理论的重要性，可以指导未来的努力设计高效 药物和生物催化剂。一种研究起源的一种策略使用酶催化的H-隧道反应是 对供体 - 受体距离敏感（dads），因此对任何可以采样H-的dads的蛋白质运动 隧道发生。在当代H-Tunnelning理论中，较重的H同位素的隧道需要一个 较短的爸爸，导致同位素速率差，从而产生动力学同位素效应（KIE）。结果，Kie是一个 爸爸的功能。因此，可以使用KIE的温度依赖性的研究来反映如何 酶动力学会影响父亲分布，因此它们是否影响酶的化学。在 在过去的二十年中，经常发现KIE与T不依赖于各种野生型酶 但是，对于不同的变体而言，t依赖于不同程度。在这些理论中，独立的KIE 由于强烈的酶有效位点压缩，已经用狭窄的爸爸来解释 效果，而变体中的强烈t依赖性kies对应于宽分布的父亲 从自然界的动态影响（部分）丧失。虽然证据支持解释 然而，被堆积起来，使用这样的Kie工具来评估这种物理起源进行催化，但是已经很热了 辩论。使用其他H转移/隧道理论对结果的模拟提出了替代解释。我们 可以测试有关KIE与dad在酶中爸爸采样的T依赖性相关性的想法 通过研究解决方案中的“简单”反应，可以通过结构来控制爸爸 效果。我们的长期目标是设计溶液中的H转移反应，以复制T依赖性的T依赖性 酶与变体中的KIE，以发现KIE观察是否是由或部分引起的。 提出的酶的耦合动力学。假设是一个更刚性的H-转移系统，具有较大的范围 人口稠密的爸爸产生了KIE的T依赖性较弱。具体目的是使用电子，空间， 溶剂和偏远的重型组振动对逐渐媒体系统刚性的效果，以调查 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 结果可以与酶的结果更直接。将确定反应的动力学 光谱镜。结果将提供有关是否有酶有效位点的论点的见解 压缩效果。该项目的另一个意义是，对 KIE的结构/溶剂与T依赖性之间的关系将打开一个新的研究方向，可以 帮助找到适当的模型来描述溶液和酶中的氢化物隧穿化学。