HOW DO ENZYMES GENERATE AND CONTROL FREE RADICALS

本申请旨在利用本项目的预付款 在目前的R29资助下。 其目的是取代这一 R01奖资助的扩展研究项目。 自由基通常被认为是高活性物质， 对细胞有害。 然而，越来越多的酶 已知使用碳基自由基来催化多种重要 代谢反应腺苷钴胺素（辅酶B12）作为一种 自由基的"掩蔽"形式，通过 辅酶钴碳键。 该基团用于除去氢原子 原子从衬底，从而激活衬底朝向 反应 我们正在研究腺苷钴胺素依赖性异构化 由谷氨酸盐催化的谷氨酸盐转化为3-甲基天冬氨酸盐， 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，这是衬底活化的关键步骤， 催化？c）酶如何控制反应性的重排 底物自由基中间体？ 当被酶结合时，组氨酸残基与钴反式配位， 轴向的钴-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 这些残留物的催化作用，我们将研究咪唑的能力， 和其它外源配体来拯救突变体中的活性， 组氨酸和天冬氨酸已被删除。 我们将决定 改变配体的pKa与拯救能力相关 酶活性 我们将完成对自由能的分析 谷氨酸盐反应的特征。 停流光谱学， 快速淬火流动技术和氚分配实验将 用于测量衬底之间的氢转移速率， 辅酶和产物，产物在酶上形成的速率，和 底物自由基重排的速率。这些测量将 提供了一个更详细的描述自由基反应比已经 可能以前。 为了检验关于原子重排的机械假说， 我们将研究底物类似物的能力， 作为替代底物和/或基于机制的抑制剂， 谷氨酸盐 的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是 用等温滴定量热法研究。 这些研究旨在 提供洞察力如何结合能可能有助于激活 辅酶对均裂。 最后，我们将继续X光检查 晶体学和蛋白质核磁共振研究来阐明这三个- 酶的三维结构。

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.