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，有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）底物活化中的关键步骤氢的去除是如何催化的？c）酶如何控制反应性底物-自由基中间体的重排，使其朝向生产性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）作为自由基的“掩蔽”形式，其通过辅酶钴-碳键的均裂而释放。该自由基用于从底物中除去氢原子，从而活化底物进行反应。我们正在研究腺苷钴胺素依赖的异构化谷氨酸3-甲基天冬氨酸，催化的谷氨酸，作为这类酶的范例。我们的目标是使用诱变来研究蛋白质如何催化辅酶的均裂和控制活性位点的自由基物种。各种活性位点突变体的动力学性质将使用快速反应技术的组合进行详细研究，并确定所选突变体的晶体结构，以便结构变化与催化变化相关。 我们还将开始研究新发现的甘氨酰自由基酶，苄基琥珀酸合成酶，这是参与各种细菌的甲苯厌氧降解的机制。该酶催化一个显著的反应-甲苯加成到富马酸酯的双键上形成（R）-苄基琥珀酸酯。它被认为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上的相似性，但是催化的化学性质非常不同。我们将研究酶的动力学性质，并试图确定和表征参与反应的各种自由基中间体。我们将探索酶的底物特异性，以尝试和识别基于机制的蛋白质抑制剂，并评估酶解毒各种芳香族化合物的潜力。

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.