HOW DO ENZYMES GENERATE AND CONTROL FREE RADICALS

酶如何产生和控制自由基

• 批准号: 6386451
• 负责人: E NEIL MARSH
• 金额: $ 23.49万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386451
• 关键词: X ray crystallography; acidity /alkalinity; active sites; chemical reaction; cobalt; cobamide; cofactor; crystallization; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; free radicals; glutamates; hydrogen ions; imidazole; ionization; isomerase; microcalorimetry; nuclear magnetic resonance spectroscopy; solutions; stop flow technique; thermodynamics; tritium

This application seeks to capitalize upon advances made in this project under the currently funded R29 award. The intention is to replace this award with an expanded research program funded by an R01 award. Free radicals are generally perceived as highly reactive species that are harmful to the cell. There is, however, a growing number of enzymes known that use carbon-based radicals to catalyze a variety of important metabolic reactions. Adenosylcobalamin (coenzyme B12) serves as a "masked" form of free radical that is liberated by homolysis of the coenzyme cobalt-carbon bond. The radical is used to remove a hydrogen atom from the substrate, thereby activating the substrate towards reaction. We are studying the adenosylcobalamin-dependent isomerization of glutamate to 3-methylaspartate, catalyzed by glutamate mutase, as a model system to investigate several fundamental aspects of enzyme- mediated radical catalysis. a) How do enzymes generate radicals? b) How is the removal of hydrogen, the key step in substrate activation, catalyzed? c) How does the enzyme control the rearrangement of reactive substrate-radical intermediates? When bound by the enzyme, a histidine residue coordinates cobalt trans- axially to the cobalt-carbon bond; the histidine, in turn, participates in a hydrogen bond with an aspartate residue. To probe the role of these residues in catalysis, we will examine the ability of imidazole and other exogenous ligands to rescue activity in mutants in which the histidine and aspartate have been deleted. We will determine whether changes the pKa of the ligand correlate with the ability to rescue enzyme activity. We will complete our analysis of the free energy profile of the glutamate mutase reaction. Stopped flow spectroscopy, rapid quenched flow techniques, and tritium partioning experiments will be used to measure the rates of hydrogen transfer between substrate, coenzyme and product, the rate of product formation on the enzyme, and the rates of substrate-radical rearrangement. These measurements will provide a more detailed description of a radical reaction than has been possible previously. To test mechanistic hypotheses concerning the rearrangement of the substrate-radical we will examine the ability of substrate analogs to function as alternative substrates and/or mechanism-based inhibitors of glutamate mutase. Thermodynamic aspects of the interactions of the protein with coenzyme, substrates and reaction intermediates will be studied by isothermal titration microcalorimetry. These studies aim to provide insight into how binding energy may contribute to activate the coenzyme towards homolysis. Finally, we will continue x-ray crystallography and protein NMR studies to elucidate the three- dimensional structure of the enzyme.

此申请旨在利用在此项目中取得的进展 在目前资助的R29奖励下。其目的是要取代这一点 由R01奖资助的扩展研究计划的奖项。 自由基通常被认为是高度活性的物种， 对细胞有害。然而，有越来越多的酶 已知使用碳基自由基催化各种重要的 代谢反应。腺苷钴胺(辅酶B12)是一种 “遮盖”形式的自由基，通过同源裂解而释放。 辅酶钴-碳键。基团用来除去一种氢 原子从基片上移出，从而激活基片朝向 反应。我们正在研究腺苷钴胺依赖的异构化 谷氨酸变位酶催化谷氨酸转化为3-甲基天冬氨酸 研究酶的几个基本方面的模型系统- 介导的自由基催化。A)酶是如何产生自由基的？B)如何 是脱氢，底物活化的关键步骤， 催化的？C)酶是如何控制反应性重排的 底物-自由基中间体？ 当被酶结合时，组氨酸残基配位钴反式- 轴向钴-碳键；组氨酸反过来参与 与天冬氨酸残基形成氢键。探讨……的作用 在这些残留物的催化作用下，我们将考察咪唑的能力 和其他外源配体来挽救突变体的活性 组氨酸和天冬氨酸已被删除。我们将确定是否 配体pKa的变化与解救能力的相关性 酶活性。我们将完成对自由能的分析 谷氨酸变位酶反应的概况。停流光谱学， 快速淬火流动技术和氚粒子实验将 用于测量衬底之间的氢转移速率， 辅酶和产物，酶上产物的形成速度，以及 底物-自由基重排的速率。这些测量结果将 提供比以往更详细的自由基反应描述 以前有可能。 来检验关于重排的机械假说 底物-我们将考察底物类似物的能力 用作替代底物和/或基于机制的抑制剂 谷氨酸变位酶。分子间相互作用的热力学问题 含有辅酶、底物和反应中间体的蛋白质 采用等温滴定微量热法进行研究。这些研究旨在 洞察结合能如何有助于激活 辅酶趋向均一化。最后，我们将继续进行x光检查。 结晶学和蛋白质核磁共振研究，以阐明三个- 酶的空间结构。