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种。然而，已知的越来越多的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作为模型系统的酶，以研究酶介导的自由基催化的几个基本方面，a)酶是如何产生自由基的？B)底物活化的关键步骤--氢的去除是如何被催化的？C)酶如何控制活性底物-自由基中间体的重排，使其成为有效的催化反应，而不是有害的副反应？ 一类重要的自由基酶使用腺苷钴胺(辅酶B12)作为自由基的“掩蔽”形式，通过辅酶钴-碳键的同解而释放出来。该自由基用来从底物上除去氢原子，从而激活底物进行反应。我们正在研究谷氨酸变位酶催化的腺苷钴胺依赖的谷氨酸异构化为3-甲基天冬氨酸，作为研究这类酶的范例。我们的目标是利用突变来研究蛋白质如何催化辅酶的同源降解和控制活性部位的自由基物种。将结合快速反应技术详细研究各种活性位点突变体的动力学性质，并确定选定突变体的晶体结构，以便结构变化与催化变化相关联。 我们还将开始研究新发现的甘氨酰自由基酶--苄基琥珀酸合成酶的机制，该酶参与了各种细菌对甲苯的厌氧降解。该酶催化甲苯与富马酸双键的加成反应生成(R)-丁二酸苄酯。它被认为与核苷酸还原酶和丙酮酸甲酸裂解酶在结构和机理上有相似之处，但催化的化学成分却非常不同。我们将研究酶的动力学性质，并尝试鉴定和表征参与反应的各种自由基中间体。我们将探索该酶的底物特异性，试图寻找该蛋白的基于机制的抑制剂，并评估该酶对各种芳香族化合物的解毒潜力。

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.