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

描述(由申请人提供)：利用缺乏电子的自由基物种的极端反应性的酶执行生物学中一些最困难的化学反应。这些酶实现的区域和立体选择性打破了长期以来认为自由基反应是非特异性的观点。这一类包括：催化DNA生物合成第一步的核糖核苷酸还原酶，阿司匹林和其他非类固醇抗炎药的靶标前列腺素H合成酶，以及催化代谢物共价键重排的辅酶B12依赖的酶家族。催化剂中常见的第一步是金属辅助生成缺电子的有机自由基。这种引发剂本身或通过二次自由基物种，促进氢原子从底物上抽离，形成底物基自由基，打开了一个新的反应通道，促进了产物自由基的重排。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，以实现高产率的高效反应。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是拟议研究的持续重点。腺苷钴胺依赖系统，特别是乙醇胺脱氨酶，已经被选中进行审查。通过脉冲电子顺磁共振和可见光/近红外吸收光谱技术，研究全酶组装和自由基对的产生、分离和稳定的机制，利用低温捕获样品和时间分辨跟踪从皮秒到几个小时的时间尺度。这些结果将被用来构建酶反应的详细分子机制。开发的洞察力和新方法将促进识别其他酶反应中的自由基中间体，为体内程序化的部位特异性自由基反应指明设计，并帮助治疗努力对抗具有生物破坏性的自由基。

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.