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）：利用缺电子自由基物种的极端反应性的酶进行生物学中一些最困难的化学反应。 这些酶所实现的区域选择性和立体选择性违背了自由基反应是非特异性的长期观念。 该类包括以下酶：核糖核苷酸还原酶（催化 DNA 生物合成中第一个独特步骤）、前列腺素 H 合酶（阿司匹林和其他非甾体抗炎药的靶标）以及辅酶 B12 依赖性酶家族（催化代谢物共价键重排。 催化中常见的主要步骤是金属辅助生成缺电子有机自由基。 该引发剂自由基本身或通过次级自由基物种，促进氢原子从底物中提取，形成基于底物的自由基，打开一个新的反应通道，促进重排为产物自由基。 一个突出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生产反应。 阐明蛋白质和辅助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。 已选择腺苷钴胺素依赖性系统，特别是乙醇胺脱氨酶进行详细检查。 全酶组装、自由基对生成、分离和稳定的机制将通过脉冲电子顺磁共振和可见/近红外吸收光谱技术，通过使用低温捕获的样品和皮秒到小时的时间尺度上的时间分辨跟踪来研究。 结果将用于构建酶反应的详细分子机制。 所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识别，指示体内程序化位点特异性自由基反应的设计，并协助对抗生物破坏性自由基的治疗工作。