HOW DO ENZYMES GENERATE AND CONTROL FREE RADICALS

酶如何产生和控制自由基

• 批准号: 2828013
• 负责人: E NEIL MARSH
• 金额: $ 24.36万
• 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/2828013
• 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.

该应用程序旨在利用该项目取得的进展 根据目前资助的 R29 奖项。 目的是取代这个 奖以及由 R01 奖资助的扩展研究计划。 自由基通常被认为是高活性物质， 对细胞有害。 然而，酶的数量却在不断增加。 已知利用碳基自由基来催化多种重要的 代谢反应。腺苷钴胺素（辅酶 B12）作为 “掩蔽”形式的自由基是通过均分解而释放的 辅酶钴碳键。 该自由基用于除去氢 原子从基板上脱落，从而激活基板 反应。 我们正在研究腺苷钴胺素依赖性异构化 在谷氨酸变位酶的催化下，谷氨酸转化为 3-甲基天冬氨酸 模型系统来研究酶的几个基本方面 介导的自由基催化。 a) 酶如何产生自由基？ b) 如何 是去除氢，这是底物活化的关键步骤， 催化？ c) 酶如何控制反应性重排 底物自由基中间体？ 当与酶结合时，组氨酸残基协调钴反式 与钴-碳键轴向；组氨酸反过来参与 与天冬氨酸残基形成氢键。 探究的作用 这些残留物的催化能力，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，其中 组氨酸和天冬氨酸已被删除。 我们将确定是否 改变配体的 pKa 与救援能力相关 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。 停流光谱法， 快速骤冷流技术和氚分配实验将 用于测量底物之间氢转移的速率， 辅酶和产物，酶上产物形成的速率，以及 底物自由基重排的速率。这些测量将 提供了比以往更详细的自由基反应描述 以前可能。 检验有关重排的机制假设 底物自由基 我们将检查底物类似物的能力 作为替代底物和/或基于机制的抑制剂 谷氨酸变位酶。 相互作用的热力学方面 蛋白质与辅酶、底物和反应中间体将 通过等温滴定微量热法进行研究。 这些研究旨在 深入了解结合能如何有助于激活 辅酶均解。 最后我们继续X光检查 晶体学和蛋白质核磁共振研究阐明了三 酶的空间结构。