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）：氢原子转移反应是催化多种重要代谢反应的一大类酶的基础，其中碳基自由基是关键中间体。从底物中自由基提取非酸性氢原子是各种不寻常和化学上困难的转化中的关键活化步骤。在许多酶中，由腺苷钴胺素或S-腺苷甲硫氨酸还原产生的5 '-脱氧腺苷自由基作为夺氢的辅因子;在其他酶中，基于蛋白质的自由基，通常是半胱氨酰自由基，作为自由基辅因子。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被稳定到显著的程度。酶产生和稳定活性自由基的机制仍然知之甚少。我们的目标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。 我们将集中在理解氢原子转移的两个自由基酶，作为模型系统。谷氨酸酯酶是腺苷钴胺素依赖性酶，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。苄基琥珀酸合成酶是一种甘氨酰自由基酶，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。我们将整合对酶和非酶模型反应的实验测量与这些反应的计算研究，以提供一个框架，在此框架内了解酶催化的氢原子转移反应和非酶反应之间的差异。 在我们将进行的实验中，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盐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合理化的线性自由能关系，如扩展的埃文斯-波兰尼方程和哈米特分析，成功地预测了大量的非酶促氢转移反应的速率基于简单的经验确定的参数。我们将测量苄基琥珀酸合成酶和谷氨酸酯酶与底物类似物反应时的氢转移速率，所述底物类似物含有能够稳定不同量的所得底物自由基的取代基。这些实验应该提供洞察力的相对重要性的双极性，空间位阻和极性效应在酶催化的氢转移反应。 公共卫生相关性：虽然活性自由基通常被认为对生物体有害，但有许多生物化学反应涉及对生命至关重要的自由基。我们正在研究利用这些自由基的酶如何控制它们，并利用它们的反应性来达到“好”的目的。更好地了解这些酶可能有助于设计治疗癌症和关节炎等疾病的药物，以及工程微生物来清理环境中的有毒化学物质。