Spectroscopic Studies of Enzyme/Substrate Complexes

酶/底物复合物的光谱研究

• 批准号: 6687833
• 负责人: GEORGE H REED
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6687833
• 关键词: Spectroscopic; Studies; Enzyme; Substrate; Complexes

DESCRIPTION (provided by applicant): The objectives of the project are to gain a better understanding of the free radical based mechanisms for enzymic catalysis and the role of the adenosyl radical in initiation of the these reactions. Free-radical reactions are of fundamental importance in biology. Although radicals that are generated adventitiously in living system are detrimentral, nature uses free radicals in order to carry out difficult steps in biosynthesis and metabolism. The adenosylcobalamin (AdoCbl)-dependent enzymes represent one famiily of enzymes that carry out free radical mediated reactions. Ethanolamine ammonia-lyase is an AdoCbl-dependent enzyme from bacteria. The enzyme catalyzes the conversion of ethanolamine to acetaldehyde and ammonia, and bacteria which contain the genes for this enzyme are able to grow on ethanolamine, obtained from the breakdown of phospholipids, as their sole source of carbon, nitrogen and energy. The enzyme is a member of a group of AdoCbl-dependent enzymes which catalyze the interchange of positions of a hydrogen atom and a substituent on adjacent carbon atoms. These enzymes use AdoCbl to initiate the radical-mediated rearrangement through homolysis of the cobalt-carbon bond of AdoCbl to give cob(ll)alamin and the reactive 5'-deoxyadenosyl radical. Some of the free radical intermediates in the reaction of substrates reach concentrations such that they can be observed by electron paramagnetic resonance (EPR) spectroscopic methods. These intermediates will be identified through their characteristic nuclear hyperfine splitting patterns in EPR spectra. Substrate analogs which lead to suicide inactivation of the enzyme by free-radicals that stray from the normal reaction pathway will be identified by spectroscopic methods and by identifiction of the reaction products. Factors leading to a greater than trillion fold acceleration in the rate of cobalt--carbon bond cleavage in enzyme-bound AdoCbl will be probed by measurement of the epimerization of a chiral 5'-deutero form of AdoCbl using nuclear magnetic resonance methods. The mechanism for the rearrangement steps in the catalytic cycle will be probed by measurement of nitrogen kinetic isotope effects for substrates using isotope ratio mass spectrometry. Genes for the enzyme from different species of bacteria will be cloned and over expressed in E. coli, and the proteins subjected to screens for crystallization.

描述（由申请人提供）：该项目的目标是更好地了解基于自由基的酶催化机制以及腺苷自由基在引发这些反应中的作用。自由基反应在生物学中具有根本重要性。尽管生命系统中偶然产生的自由基是有害的，但大自然利用自由基来执行生物合成和代谢中的困难步骤。腺苷钴胺素 (AdoCbl) 依赖性酶代表了一类进行自由基介导反应的酶。乙醇胺氨裂解酶是一种来自细菌的 AdoCbl 依赖性酶。该酶催化乙醇胺转化为乙醛和氨，含有该酶基因的细菌能够依靠从磷脂分解中获得的乙醇胺生长，作为其碳、氮和能量的唯一来源。该酶是一组 AdoCbl 依赖性酶的成员，该酶催化相邻碳原子上的氢原子和取代基的位置互换。这些酶使用 AdoCbl 通过 AdoCbl 的钴-碳键均裂来引发自由基介导的重排，产生钴 (II) 胺和反应性 5'-脱氧腺苷自由基。底物反应中的一些自由基中间体达到可以通过电子顺磁共振（EPR）光谱方法观察的浓度。这些中间体将通过 EPR 光谱中其特有的核超精细分裂模式进行识别。通过偏离正常反应途径的自由基导致酶自杀性失活的底物类似物将通过光谱方法和反应产物的鉴定来鉴定。将通过使用核磁共振方法测量 AdoCbl 的手性 5'-氘代形式的差向异构化来探究导致酶结合的 AdoCbl 中钴-碳键断裂速率加速超过万亿倍的因素。将通过使用同位素比质谱测量底物的氮动力学同位素效应来探讨催化循环中重排步骤的机制。来自不同种类细菌的酶基因将被克隆并在大肠杆菌中过度表达，并且蛋白质将接受结晶筛选。