Understanding hydrogen atom transfer reactions in enzymes

描述（由申请人提供）：氢原子转移反应是一大类酶的基础，它们催化多种重要的代谢反应，其中碳基自由基是关键中间体。从底物中提取非酸性氢原子的自由基是各种不寻常的和化学上困难的转化的关键激活步骤。在许多酶中，由腺苷钴胺素或s -腺苷蛋氨酸还原产生的5'-脱氧腺苷自由基作为提取氢的辅助因子；在其他情况下，以蛋白质为基础的自由基，通常是半胱氨酸基自由基，作为自由基辅助因子。与自由基在自由溶液中的反应性相比，酶中产生的自由基似乎得到了显著的稳定。酶产生和稳定活性自由基的机制仍然知之甚少。我们的目的是详细研究氢原子转移，底物活化的关键步骤，是如何催化的，目的是阐明酶催化自由基反应的一般原理。我们将重点了解作为模型系统的两个自由基酶中的氢原子转移。谷氨酸变异酶是一种依赖腺苷钴胺的酶，其中氢原子转移用于产生底物自由基，然后进行碳骨架重排。苄基琥珀酸合成酶是一种甘酰基自由基酶，其中氢从甲苯转移到活性位点半胱氨酸是一个关键的机制特征。我们将把酶和非酶模型反应的实验测量与这些反应的计算研究结合起来，为理解酶催化的氢原子转移反应和非酶反应之间的差异提供一个框架。我们将进行的实验包括动力学同位素效应测量，旨在确定迁移氢原子的量子隧穿在谷氨酸突变酶催化的反应中有多大程度的重要性。我们将把这些结果与突变酶和模型非酶B12反应的结果进行比较。通过使用最先进的QM/MM计算方法对酶和非酶反应进行建模，我们将深入了解酶的催化作用，特别是酶是否实际上增加了反应中的量子隧穿量以增强催化作用。我们将测试酶催化的氢原子转移反应是否可以通过线性自由能关系（如扩展的Evans-Polanyi方程和Hammett分析）来合理解释，这些关系成功地预测了基于简单经验确定的参数的大量非酶氢转移反应的速率。我们将测量氢的转移速率在苯琥珀酸合酶和谷氨酸变异酶反应时，底物类似物含有取代基能够稳定所产生的底物自由基的不同量。这些实验应该为酶催化氢转移反应中焓、位和极性效应的相对重要性提供见解。

DESCRIPTION (provided by applicant): Hydrogen atom transfer reactions are fundamental to a large class of enzymes that catalyze a diverse array of important metabolic reactions in which carbon-based radicals are key intermediates. Radical abstraction of a non-acidic hydrogen atom from the substrate is the key activation step in a variety of unusual and chemically difficult transformations. In many enzymes 5'-deoxyadenosyl radical, generated from either adenosylcobalamin or reduction of S-adenosylmethionine, serves as the cofactor for hydrogen abstraction; in others a protein-based radical, often a cysteinyl radical, serves as the radical cofactor. Compared to their reactivity in free solution, free radicals generated in enzymes appear to be stabilized to a remarkable degree. The mechanisms by which enzymes generate and stabilize reactive free radicals remain poorly understood. Our aim is to investigate in detail how hydrogen atom transfer, the key step in substrate activation, is catalyzed with the goal of illuminating the general principles by which enzymes catalyze radical reactions. We will focus on understanding hydrogen atom transfer in two radical enzymes that serve as model systems. Glutamate mutase is an adenosylcobalamin-dependent enzyme in which hydrogen atom transfer serves to generate a substrate radical that then undergoes a carbon skeleton rearrangement. Benzylsuccinate synthase is a glycyl-radical enzyme in which hydrogen transfer from toluene to an active site cysteine is a key mechanistic feature. We will integrate experimental measurements on enzymes and non-enzymatic model reactions with computational studies of these reactions to provide a framework within which to understand the differences between enzyme-catalyzed hydrogen atom transfer reactions and non-enzymatic reactions. Among the experiments we will conduct are kinetic isotope effect measurements that aim to determine to what extent quantum tunneling of the migrating hydrogen atom is important in the reaction catalyzed by glutamate mutase. We will compare these results with those obtained for mutant enzymes and model, non- enzymatic B12 reactions. By modeling both enzymatic and non-enzymatic reactions using state-of-the-art QM/MM computational methods we will gain insights into catalysis by the enzyme, and, in particular, whether the enzyme actually increases the amount of quantum tunneling in a reaction to enhance catalysis. We will test whether enzyme-catalyzed hydrogen atom transfer reactions can rationalized by linear free energy relationships such as the extended Evans-Polanyi equation and Hammett analysis that successfully predict the rates of a large number of non-enzymatic hydrogen transfer reactions based on simple empirically determined parameters. We will measure the rates of hydrogen transfer in benzylsuccinate synthase and glutamate mutase when reacted with substrate analogs containing substituents capable of stabilizing the resultant substrate radicals by different amounts. These experiments should provide insights into the relative importance of enthalpic, steric and polar effects in enzyme-catalyzed hydrogen transfer reactions. PUBLIC HEALTH RELEVANCE: Although reactive free radicals are generally perceived as harmful to living organisms, there are many biochemical reactions that involve free radicals that are essential for life. We are studying how the enzymes that use these free radicals control them and harness their reactivity for "good" purposes. A better understanding of these enzymes may help in the design of drugs to fight diseases such as cancer and arthritis, and in engineering micro-organisms to clean up toxic chemicals in the environment.