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

描述（由申请人提供）：利用缺电子自由基物种的极端反应性进行生物学中一些最困难的化学反应的酶。 这些酶所实现的区域和立体选择性挑战了长期以来认为自由基反应是非特异性的想法。 这类酶包括：核糖核苷酸还原酶，催化DNA生物合成的第一个独特步骤;前列腺素H合酶，阿司匹林和其他非甾体抗炎药的靶点;辅酶B12依赖性酶家族，催化代谢产物共价键重排。 在催化中的共同的主要步骤是金属辅助的缺电子有机自由基的产生。 这种引发剂自由基，无论是通过自身或通过第二自由基物种，促进氢原子从基板抽象，以形成基于基板的自由基，打开一个新的反应通道，促进重排的产物自由基。 一个突出的问题是如何使自由基对稳定以防止快速重组，从而以高产率实现生产性反应。 阐明蛋白质和辅因子如何引导自由基稳定和随后的底物自由基重排的基本原理将成为拟议研究的持续重点。 腺苷钴胺素依赖性系统，特别是乙醇胺脱氨酶，已被选定进行审查。 全酶组装，和自由基对的产生，分离和稳定的机制将研究通过脉冲电子顺磁共振和可见/近红外吸收光谱技术，通过使用cryotraped样品和时间分辨跟踪的时间尺度从皮秒到小时。 这些结果将用于构建酶反应的详细分子机理。 开发的见解和新方法将促进其他酶反应中自由基中间体的鉴定，指示体内程序化位点特异性自由基反应的设计，并协助治疗工作以对抗生物破坏性自由基。

DESCRIPTION (provided by applicant): 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-inflammatory 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.