How do Enzymes Generate and Control Free Radicals?

描述（由申请人提供）： 自由基通常被认为是对细胞有害的高活性物质。然而，越来越多的已知酶使用碳基自由基来催化各种重要的代谢反应。我们正在研究两种以完全不同的方式使用自由基的酶作为模型系统，以研究酶介导的自由基催化的几个基本方面：a) 酶如何产生自由基？ b) 底物活化的关键步骤——氢的去除是如何被催化的？ c) 酶如何控制反应性底物自由基中间体的重排以实现高效催化而不是有害的副反应？ 一类重要的自由基酶使用腺苷钴胺素（辅酶 B12）作为“掩蔽”形式的自由基，通过辅酶钴碳键的均裂释放自由基。该自由基用于从基材上除去氢原子，从而激活基材进行反应。我们正在研究谷氨酸变位酶催化的腺苷钴胺素依赖性谷氨酸异构化为 3-甲基天冬氨酸，作为此类酶的范例。我们的目标是利用诱变来研究蛋白质如何催化辅酶均裂并控制活性位点的自由基种类。将使用快速反应技术的组合详细检查各种活性位点突变体的动力学特性，并将确定所选突变体的晶体结构，以便结构的变化可以与催化的变化相关联。 我们还将开始研究新发现的甘氨酰自由基酶——苄基琥珀酸合酶的机制，该酶参与多种细菌对甲苯的厌氧降解。该酶催化一种显着的反应 - 将甲苯加成到富马酸的双键上，形成 (R)-苄基琥珀酸。据信，它与核糖核苷酸还原酶和丙酮酸甲酸裂解酶具有结构和机制相似性，但催化的化学性质却截然不同。我们将检查酶的动力学特性，并尝试识别和表征反应中涉及的各种自由基中间体。我们将探索该酶的底物特异性，以尝试和识别基于机制的蛋白质抑制剂，并评估该酶解毒各种芳香化合物的潜力。

DESCRIPTION (provided by applicant): Free radicals are generally perceived as highly reactive species that are harmful to the cell. There are, however, a growing number of enzymes known that use carbon-based radicals to catalyze a variety of important metabolic reactions. We are studying two enzymes that use free radicals in quite different ways as model systems to investigate several fundamental aspects of enzyme-mediated radical catalysis, a) How do enzymes generate radicals? b) How is the removal of hydrogen, key step in substrate activation, catalyzed? c) How do enzymes control the rearrangement of reactive substrate-radical intermediates towards productive catalysis rather than harmful side reactions? One important class of radical enzymes uses adenosylcobalamin (coenzyme B12)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 paradigm for this class of enzymes. We aim to use mutagenesis to investigate how the protein catalyzes homolysis of the coenzyme and controls radical species at the active site. The kinetic properties of various active site mutants will be examined in detail using a combination of rapid-reaction techniques and the crystal structures of selected mutants will be determined so that changes in structure can be correlated with changes in catalysis. We will also start to investigate the mechanism of the newly discovered glycyl radical enzyme, benzylsuccinate synthase, which is involved in the anaerobic degradation of toluene by various bacteria. The enzyme catalyzes a remarkable reaction - addition of toluene to the double bond of fumarate to form (R)-benzylsuccinate. It is believed to share structural and mechanistic similarities with ribonucleotide reductase and pyruvate formate-lyase, however the chemistry catalyzed is very different. We will examine the kinetic properties of the enzyme and attempt to identify and characterize various radical intermediates involved in the reaction. We will explore the substrate specificity of the enzyme to try and identify mechanism-based inhibitors of the protein and to evaluate the potential of the enzyme to detoxify various aromatic compounds.