Co(II)-Radical Pair Dynamics in B12 Enzyme Catalysis

利用缺乏电子的自由基物种的极端反应性的酶执行一些 生物学中最难的化学反应。这些酶实现的区域和立体选择性 挑战了长期以来认为激进反应是非特异性的观点。本课程包括以下内容： 核苷酸还原酶，催化DNA生物合成的第一个独特步骤，前列腺素H- 合成酶，阿司匹林和其他非类固醇抗炎症药物的靶标，以及辅酶家族 依赖B12的酶，催化代谢物共价键重排。共同的初选 催化剂中的步骤是金属辅助生成缺电子的有机自由基。此发起人 自由基本身或通过次级自由基物种，促进氢原子从 底物形成基于底物的自由基，打开一个新的反应通道，促进重排 一个激进的产品。一个悬而未决的问题是如何稳定自由基对以防止快速重组 实现高产率的生产性反应。阐明蛋白质和辅因子引导的基本原理 自由基稳定和随之而来的底物自由基重排将是拟议的持续重点 学习。腺苷钴胺依赖系统，特别是乙醇胺脱氨酶，一直是 被选中接受审查。全酶组装和自由基对的产生、分离和 稳定性将通过脉冲电子顺磁共振和可见光/近红外光谱技术进行研究。 利用深冷样品的红外吸收光谱和时间尺度上的时间分辨跟踪 从皮秒到几个小时不等。这些结果将被用来构建详细的分子机制 用于酶反应。所开发的洞察力和新方法将促进激进分子的识别 其他酶反应中的中间体，表明设计为程序化的部位特异性自由基反应 活体，并协助治疗努力，以对抗具有生物破坏性的自由基。

Enzymes that harness the extreme reactivity of electron-deficient free radical species carry out some of the most difficult chemical reactions in biology. The regio- and stereo-selectivity achieved by these enzymes defies long-held ideas that radical reactions are non-specific. This class includes the following: ribonucleotide reductases, which catalyze the first unique step in DNA biosynthesis, prostaglandin H- synthase, the target of aspirin and other non-steroidal anti-infiamatory drugs, and the family of coenzyme B12-dependent enzymes, which catalyze metabolite covalent bond rearrangements. The common primary step in the catalyses is metal-assisted generation of an electron-deficient organic radical. This initiator radical, either by itself or through secondary radical species, promotes hydrogen atom abstraction from the substrate to form a substrate-based radical, opening a new reaction channel that facilitates rearrangement to a product radical. An outstanding issue is how the radical pair is stabilized against rapid recombination to achieve productive reaction in high yield. Elucidating the basic principles of how protein and cofactors guide radical stabilization and ensuing substrate radical rearrangement will be sustained focuses of the proposed studies. The adenosylcobalamin-dependent systems, and ethanolamine deaminase specifically, have been selected for scrutiny. The mechanisms of holoenzyme assembly, and radical pair generation, separation and stabilization will be studied by techniques of pulsed-electron paramagnetic resonance and visible/near- infrared absorption spectroscopy by using cryotrapped samples and time-resolved tracking on time scales ranging from picoseconds to hours. The results will be used to construct a detailed molecular mechanism for the enzyme reactions. The insights and novel methods developed will promote identification of radical intermediates in other enzyme reactions, indicate designs for programmed site-specific radical reactions in vivo, and assist therapeutic efforts to combat biologically-destructive free radicals.