Understanding hydrogen atom transfer reactions in enzymes

了解酶中的氢原子转移反应

• 批准号: 7863509
• 负责人: E NEIL MARSH
• 金额: $ 27.57万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7863509
• 关键词: Active Sites; Arthritis; Arts; Behavior; Biochemical; Biochemical Reaction; Biological Models; Carbon; Catalysis; Coenzymes; Computer Analysis; Computer Simulation; Computers; Computing Methodologies; Cysteine; DNA Sequence Rearrangement; Deuterium; Disease; Drug Design; Electrostatics; Engineering; Environment; Enzymes; Equation; Free Energy; Free Radicals; Goals; Hydrogen; Isotopes; Kinetics; Life; Malignant Neoplasms; Measurement; Measures; Metabolic; Methods; Modeling; Mutate; Mutation; Organism; Poison; Property; Proteins; Quantum Mechanics; Reaction; Relative (related person); Site-Directed Mutagenesis; Skeleton; Solutions; Solvents; Structure; Temperature; Testing; Toluene; analog; base; benzylsuccinate synthase; chemical reaction; cobamamide; cofactor; computer studies; density; enthalpy; enzyme mechanism; enzyme model; fighting; insight; methylaspartate mutase; molecular mechanics; mutant; public health relevance; quantum; research study; small molecule; theories

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.

描述（由申请人提供）：氢原子转移反应对大量酶的基础是催化各种重要代谢反应的各种酶，在这些酶中，基于碳的自由基是关键的中间体。从底物的非酸性氢原子的根本抽象是各种不寻常且化学困难的转化中的关键激活步骤。在许多酶5'-脱氧腺苷自由基中，是由腺苷胆汁素或S-腺苷硫氨酸还原产生的，是氢抽象的辅助因子。在其他情况下，基于蛋白质的自由基，通常是半胱氨酸自由基，用作自由基辅助因子。与它们在自由溶液中的反应性相比，在酶中产生的自由基似乎在显着程度上稳定。酶产生和稳定反应性自由基的机制仍然鲜为人知。我们的目的是详细研究氢原子转移是如何催化底物激活的关键步骤，目的是阐明酶催化自由基反应的一般原理。 我们将专注于理解两种用作模型系统的自由基酶中的氢原子转移。谷氨酸突变酶是一种依赖腺苷的酶依赖性酶，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。苄基核酸合酶是一种糖基 - 自由基酶，其中氢从甲苯转移到活性位点半胱氨酸是关键机械特征。我们将在酶和非酶模型反应上的实验测量与这些反应的计算研究相结合，以提供一个框架，以了解酶催化的氢原子转移反应与非酶反应之间的差异。 在实验中，我们将进行的是动力学同位素效应测量值，旨在确定迁移氢原子的量子隧穿在多大程度上在谷氨酸突变酶催化的反应中很重要。我们将将这些结果与突变酶和非酶促B12反应获得的结果进行比较。通过使用最先进的QM/MM计算方法对酶促和非酶促反应进行建模，我们将通过酶来洞悉酶对催化的见解，尤其是酶是否真的增加了反应中量子隧道的量是否增加增强催化。 我们将测试酶催化的氢原子转移反应是否可以通过线性的自由能关系（例如扩展的Evans-Polanyi方程和Hammett分析）合理化，从而成功地预测了基于简单经验确定的大量非酶氢转移反应的速率参数。当与含有能够以不同量稳定所得底物自由基稳定所得的底物自由基的底物类似物反应时，我们将测量苄基共酶合酶和谷氨酸突变酶的氢转移速率。这些实验应提供有关焓，空间和极性影响在酶催化的氢转移反应中的相对重要性的见解。 公共卫生相关性：尽管反应性自由基通常被认为对生物有害，但许多生化反应涉及对生命必不可少的自由基。我们正在研究使用这些自由基的酶如何控制它们并利用其反应性以“良好”目的。更好地了解这些酶可能有助于设计与癌症和关节炎等疾病以及工程微生物的疾病的设计，以清理环境中的有毒化学物质。