HOW DO ENZYMES GENERATE AND CONTROL FREE RADICALS

酶如何产生和控制自由基

基本信息

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
项目类别：
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

项目摘要

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.

该申请旨在利用该项目的进步根据目前资助的R29奖。目的是替换由R01奖项资助的扩展研究计划的奖项。自由基通常被认为是高反应性的物种对细胞有害。但是，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。腺苷胆素（辅酶B12）用作A “蒙面”的自由基形式，通过对辅酶钴碳键。自由基用于去除氢原子来自底物，从而激活底物朝向反应。我们正在研究腺基呼吸蛋白依赖性异构化谷氨酸到3-甲基天冬氨酸，由谷氨酸突变酶催化为A 模型系统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，这是底物激活的关键步骤，催化？ c）酶如何控制反应性的重排底物 - 激进的中间体？当酶约束时，组氨酸残基坐标钴trans- 轴向到钴碳键；组氨酸反过来参与在与天冬氨酸残基的氢键中。探究这些残留在催化中，我们将检查咪唑的能力和其他外源配体以拯救突变体中的活动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与营救能力相关酶活性。我们将完成对自由能的分析谷氨酸突变酶反应的谱。停止流量光谱，快速淬火流动技术和trium cotition实验将用于测量底物之间的氢转移速率，辅酶和产品，酶上的产品形成速率，以及底物 - 自由基重排的速度。这些测量结果提供对根本反应的更详细的描述以前可能。测试有关重新排列的机械假设底物 - 激进分子我们将检查底物类似物的能力起作用的替代基板和/或基于机制的抑制剂的功能谷氨酸突变酶。相互作用的热力学方面与辅酶，底物和反应中间体的蛋白质将是通过等温滴定微钙化法进行了研究。这些研究旨在提供有关结合能如何有助于激活的洞察力辅酶朝向同型。最后，我们将继续X射线晶体学和蛋白质NMR研究，以阐明三个酶的尺寸结构。