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

B12 酶催化中的 Co(II)-自由基对动力学

基本信息

批准号：
7163573
负责人：
KURT WARNCKE
金额：
$ 23.88万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
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/7163573
关键词：
Address Amines Apoenzymes Aspirin Binding Biological Biology Carbon Catalysis Cell Nucleus Class Classification Cobalamin Cobalt Coenzymes Complement component C1s Coupled Coupling DNA Sequence Rearrangement DNA biosynthesis Databases Deoxyadenosines Detection Development Electron Spin Resonance Spectroscopy Electrons Enzymes Equilibrium Ethanolamine Ammonia-Lyase Event Family Free Energy Free Radicals Frequencies Generations Genetic Recombination Goals Holoenzymes Hour Hydrogen In Situ Individual Isotopes Kinetics Lasers Lead Ligands Maps Measurement Measures Mediating Metals Methodology Methods Modeling Molecular Nitrogen Nuclear Object Attachment Optics Pharmaceutical Preparations Physiologic pulse Positioning Attribute Principal Investigator Prostaglandin-Endoperoxide Synthase Proteins Pulse taking Range Rate Reaction Research Rest Ribonucleotide Reductase Sampling Site Spectrum Analysis Staging Step Tests Structure System Techniques Testing Therapeutic Thermodynamics Time Work absorption analog base chemical reaction cob(II)alamin cobamamide coenzyme B cofactor comparative corrin covalent bond density deoxyadenosine design enzyme mechanism enzyme substrate analog flash photolysis in vivo insight metalloenzyme migration novel photolysis programs protein structure

项目摘要

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-infiamatory 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.

利用缺电子自由基物种的极端反应性的酶可以完成一些任务生物学中最困难的化学反应。这些酶实现的区域选择性和立体选择性颠覆了人们长期以来认为激进反应是非特异性的观念。该类包括以下内容：核糖核苷酸还原酶，催化 DNA 生物合成的第一个独特步骤，前列腺素 H- 合成酶，阿司匹林和其他非甾体抗炎药的靶标，以及辅酶家族 B12 依赖性酶，催化代谢物共价键重排。共同初级催化中的一步是金属辅助生成缺电子有机自由基。这位发起人自由基本身或通过次级自由基物种促进氢原子从底物形成基于底物的自由基，打开新的反应通道，促进重排产品激进。一个突出的问题是自由基对如何稳定以防止快速重组实现高收率的高效反应。阐明蛋白质和辅因子如何指导的基本原理自由基稳定和随后的底物自由基重排将是拟议的持续焦点研究。腺苷钴胺依赖性系统，特别是乙醇胺脱氨酶，已被选定进行审查。全酶组装、自由基对产生、分离和分离的机制将通过脉冲电子顺磁共振和可见光/近光技术来研究稳定性使用冷冻样品和时间尺度上的时间分辨跟踪进行红外吸收光谱范围从皮秒到小时。结果将用于构建详细的分子机制用于酶反应。所开发的见解和新颖方法将促进激进分子的识别其他酶反应中的中间体，表明了程序化位点特异性自由基反应的设计体内，并协助治疗努力对抗具有生物破坏性的自由基。