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: 2856844
• 负责人: KURT WARNCKE
• 金额: $ 13.65万
• 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/2856844
• 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.

一种新兴的酶，利用极端的反应性， 缺电子自由基物种进行一些最 生物学中困难的化学反应最近已被确定。 这些酶实现的区域和立体选择性挑战了长时间的限制。 认为激进反应是非特异性的。 这一类别包括 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，靶点 阿司匹林和其他非甾体抗炎药， B12辅酶依赖性酶家族，催化代谢产物 共价键重排 催化反应中常见的第一步 是电子中心或金属辅助产生的缺电子 有机自由基 该引发剂自由基从 底物形成基自由基，开启新的反应 促进重排为产物自由基的通道。 一 具有挑战性的问题是如何使自由基稳定以防止重组 金属。这与电荷分离反应类似 在光合和呼吸生物能复合体中， 已经建立了基本的性能原则。然而，在这方面， 不像简单的电子-空穴分离， 弱相互作用的氧化还原位点，金属自由基分离涉及大- 重核和相对强电子的尺度运动 交互. 阐明了蛋白质和 辅因子引导自由基稳定化， 重新安排将是拟议研究的持续重点。 的 腺苷钴胺素依赖系统和乙醇胺脱氨酶 特别是，由于均裂断裂， 形成CoII金属自由基和5 '- 腺苷引发剂自由基可由可见激光脉冲触发， 允许自由基对状态的相干制备， 催化条件 自由基对分离和基底自由基 重排将跟踪脉冲EPR的时间分辨技术 谱动态电子自旋-自旋与电子核超精细 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 其它酶中瞬时自由基中间体鉴定 反应，表明设计程序化的位点特异性自由基 体内反应，并协助治疗努力，以打击 具有生物破坏性的自由基