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

B12 酶催化中的 Co(II)-自由基对动力学

• 批准号: 6581018
• 负责人: KURT WARNCKE
• 金额: $ 30.25万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6581018
• 关键词: catalyst; cobalamin; cobalt; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; flash photolysis; free radicals; infrared spectrometry; metalloenzyme; vitamin B12 coenzyme

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.

描述（由申请人提供）：利用电子缺陷自由基物种极端反应性的酶进行生物学中最困难的化学反应。 这些酶实现的区域和立体观念违反了根本反应是非特异性的长期观念。 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. Catalyses的常见主要步骤是金属辅助产生电子缺陷的有机自由基。 该引发剂自由基本身或通过二级自由基物种促进了从底物中促进氢原子的抽象，从而形成了基于底物的自由基，开了一个新的反应通道，该通道促进了将重排向产物自由基的重排。 一个杰出的问题是，如何针对快速重组稳定自由基对以高产量实现生产反应。 阐明蛋白质和辅助因子如何指导自由基稳定和随之而来的底物自由基重排的基本原理将是拟议研究的持续重点。 已经选择了依赖腺苷的白氧化胺依赖性系统和乙醇胺脱氨酶进行审查。 全酶组装的机制以及激进的成对产生，分离和稳定将通过脉冲电子顺磁分辨率和可见/近红外吸收光谱的技术研究，并使用冻结的样品和时间分解的样品和时间范围从picoseconds到小时到小时。 结果将用于构建用于酶反应的详细分子机制。 开发的洞察力和新方法将促进对其他酶反应中自由基中间体的鉴定，这表明了体内编程的位点特异性自由基反应的设计，并协助治疗性努力以对抗生物造成的自由基。