Understanding hydrogen atom transfer reactions in enzymes

描述(申请人提供)：氢原子转移反应是一大类酶的基础，这些酶催化一系列重要的代谢反应，其中碳基自由基是关键的中间体。从底物上提取非酸性氢原子是各种不寻常的和化学上困难的转化中的关键活化步骤。在许多酶中，由腺苷钴胺或S-腺苷蛋氨酸还原产生的5‘-脱氧腺苷自由基是抽氢的辅助因子；在另一些酶中，一个基于蛋白质的自由基(通常是半胱氨基)充当自由基辅助因子。与它们在自由溶液中的反应性相比，酶中产生的自由基似乎稳定到了显著的程度。酶产生和稳定反应性自由基的机制仍然知之甚少。我们的目标是详细研究底物活化的关键步骤氢原子转移是如何被催化的，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种自由基酶中的氢原子转移。谷氨酸变位酶是一种腺苷钴胺依赖的酶，在该酶中，氢原子转移产生底物自由基，然后进行碳骨架重排。丁二酸苄酯合成酶是一种甘氨酸基自由基酶，其关键的机制是将氢从甲苯转移到半胱氨酸的活性部位。我们将把对酶和非酶模型反应的实验测量与对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区别提供一个框架。我们将进行的实验包括动力学同位素效应测量，旨在确定迁移中的氢原子的量子隧道在谷氨酸变位酶催化的反应中的重要程度。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计算方法对酶反应和非酶反应进行建模，我们将深入了解酶的催化作用，特别是酶是否确实增加了反应中的量子隧道量以增强催化作用。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来合理化，例如扩展的Evans-Polanyi方程和Hammett分析，这些分析基于简单的经验确定的参数成功地预测了大量非酶氢转移反应的速率。我们将测量琥珀酸苄酯合成酶和谷氨酸变位酶与底物类似物反应时的氢转移速率，底物类似物含有能够稳定生成的底物自由基不同量的取代基。这些实验应该有助于深入了解在酶催化的氢转移反应中，焓效应、空间效应和极性效应的相对重要性。 与公共健康相关：尽管反应性自由基通常被认为对活着的有机体有害，但有许多生化反应涉及对生命至关重要的自由基。我们正在研究使用这些自由基的酶如何控制它们，并利用它们的反应性达到“好”的目的。更好地了解这些酶可能有助于设计抗击癌症和关节炎等疾病的药物，并帮助设计微生物来清除环境中的有毒化学物质。

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.