COBALT(II)-RADICAL PAIR DYNAMICS IN B12 ENZYME CATALYSIS

B12 酶催化中的钴 (II)-自由基对动力学

• 批准号: 2703673
• 负责人: KURT WARNCKE
• 金额: $ 13.25万
• 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 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2703673
• 关键词: cobalamin; cobalt; electron spin resonance spectroscopy; enzyme mechanism; enzyme structure; enzyme substrate; vitamin B12 coenzyme

An emergent class of enzymes that harness the extreme reactivity of electron-deficient free radical species to carry out some of the most difficult chemical reactions in biology has been recently identified. 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 B12 coenzyme-dependent enzymes, which catalyze metabolite covalent bond rearrangements. The common primary step in the catalysis is metallocenter- or metal-assisted generation of an electron-deficient organic radical. This initiator radical abstracts a hydrogen atom from the substrate to form a substrate-based radical, opening a new reaction channel that facilitates rearrangement to a product radical. A challenging issue is how the radical is stabilized against recombination with the metal. There are analogies with the charge separation reactions in photosynthetic and respiratory bioenergetic complexes, for which the rudimentary performance principles have been established. However, unlike simple electron-hole separation by electron tunneling among weakly interacting redox sites, metal-radical separations involve large- scale movement of heavy nuclei and relatively strong electronic interactions. 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 because homolytic cleavage of the cobalt-carbon bond to form the CoII metalloradical and 5'- adenosyl initiator radical can be triggered by a visible laser pulse, allowing the coherent preparation of the radical pair state under catalytic conditions. The radical pair separation and substrate radical rearrangement will be tracked by time-resolved techniques of pulsed-EPR spectroscopy. Dynamic electron spin-spin and electron-nuclear hyperfine interactions will be measured and used to construct a detailed molecular mechanism. The insights and techniques developed will promote identification of transient radical intermediates in other enzyme reactions, indicate designs for programmed site-specific radicals reactions in vivo, and assist therapeutic efforts to combat biologically-destructive free radicals.

一类新兴的酶，利用了极端反应性 缺电子自由基物种进行一些最 最近发现了生物学中困难的化学反应。 这些酶所实现的区域选择性和立体选择性挑战了长期的研究 认为激进反应是非特异性的。 该类包括 以下：核糖核苷酸还原酶，催化第一个 DNA 生物合成中的独特步骤，前列腺素 H 合酶，目标 阿司匹林和其他非甾体类抗炎药，以及 B12 辅酶依赖性酶家族，可催化​​代谢物 共价键重排。 催化中常见的初级步骤 是缺电子的金属中心或金属辅助生成 有机自由基。 该引发剂自由基从 底物形成基于底物的自由基，开启新的反应 促进产品基团重新排列的渠道。 一个 具有挑战性的问题是如何稳定自由基以防止重组 与金属。与电荷分离反应有类似之处 在光合作用和呼吸生物能复合物中， 基本的绩效原则已经建立。然而， 与通过电子隧道效应实现的简单电子空穴分离不同 弱相互作用的氧化还原位点，金属自由基分离涉及大量 重核和相对强电子的尺度运动 互动。 阐明蛋白质如何作用的基本原理 辅助因子引导自由基稳定和随后的底物自由基 重新安排将是拟议研究的持续重点。 这 腺苷钴胺依赖性系统和乙醇胺脱氨酶 具体来说，已被选择进行审查，因为均裂 钴-碳键形成 CoII 金属自由基和 5'- 腺苷引发自由基可以由可见激光脉冲触发， 允许自由基对态的相干制备 催化条件。 自由基对分离和底物自由基 重排将通过脉冲 EPR 的时间分辨技术进行跟踪 光谱学。动态电子自旋-自旋和电子-核超精细 相互作用将被测量并用于构建详细的分子 机制。 所开发的见解和技术将促进 其他酶中瞬时自由基中间体的鉴定 反应，表明编程位点特异性自由基的设计 体内反应，并协助治疗努力对抗 具有生物破坏性的自由基。