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.

项目概要 最近提出的与酶反应化学耦合的蛋白质动力学提出了一种新的方法 酶促速率加速的可能起源。如果有的话，找到催化中的这种物理作用是很重要的 酶催化理论的发展具有重要意义，可以指导未来设计高效酶的努力 药物和生物催化剂。研究起源的一种策略是使用酶催化的氢隧道反应，该反应是 对供体-受体距离 (DAD) 敏感，因此对任何可以对 DAD 进行 H- 采样的蛋白质运动敏感 发生隧道效应。在当代 H 隧道理论中，较重 H 同位素的隧道需要 较短的 DAD，导致同位素速率差异，从而产生动力同位素效应 (KIE)。因此，KIE 是一个 DAD 的功能。因此，对 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，从而影响酶的化学性质。超过 在过去的二十年里，人们经常发现 KIE 与多种野生型酶不依赖于 T 但对于不同的变体会不同程度地依赖于 T。在这些理论中，独立于 T 的 KIE 由于强烈的酶活性位点压缩，DAD 分布较窄，这已被解释为 效应，而变体中强烈 T 依赖的 KIE 对应于广泛分布的 DAD 来自自然动力效应的（部分）损失。虽然出现了支持这些解释的证据 然而，使用此类 KIE 工具来评估催化的物理起源已成为热门话题。 争论了。使用其他氢传输/隧道理论对结果进行的模拟提出了替代的解释。我们 考虑到关于 KIE 的 T 依赖性与酶中 DAD 采样的相关性的想法可以得到测试 通过研究溶液中的“更简单”反应，DAD 可以通过结构和溶剂来控制 影响。我们的长期目标是设计溶液中的氢转移反应，以复制 T 依赖性 酶与变体中的 KIE，以确定 KIE 观察结果是否是由酶引起或部分引起的 提出了酶的耦合动力学。假设是更严格的氢转移系统具有更小的范围 密集的 DAD 导致 KIE 的 T 依赖性较弱。具体目标是利用电子、空间、 溶剂和远程重基团振动效应逐渐调节系统刚性以研究 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。将确定反应动力学 光谱上。结果将有助于深入了解是否存在酶活性位点的争论 压缩效果。该项目的另一个意义在于，前所未有地系统研究了 KIE 的结构/溶剂与 T 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道化学。