Spectroscopic Studies of Enzyme/Substrate Complexes

酶/底物复合物的光谱研究

基本信息

批准号：
7336199
负责人：
GEORGE H REED
金额：
$ 10.77万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-01-01 至 2007-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7336199
关键词：
5&apos -deoxyadenosine Acceleration Acetaldehyde Active Sites Aldehydes Ammonia Anabolism Bacteria Binding Biology Carbon Catalysis Characteristics Cleaved cell Cobalt Complex Coupled Crystallization DNA Sequence Rearrangement Electron Nuclear Double Resonance Electron Spin Resonance Spectroscopy Enzyme Gene Enzymes Escherichia coli Proteins Ethanolamine Ammonia-Lyase Ethanolamines Ethylenediamine Ethylenediamines Free Radicals Hybrids Hydrogen Hydroxyl Radical Investigation Isopropanol Isotopes Ketones Kinetics Lead Mass Spectrum Analysis Measurement Measures Mediating Mercaptoethylamine Mercaptoethylamines Metabolism Methods Molecular Monitor Natural regeneration Nature Nitrogen Nuclear Nuclear Magnetic Resonance Object Attachment Organism Oxygen Pathway interactions Pattern Personal Satisfaction Phospholipids Positioning Attribute Process Propanolamines Property Proteins Rate Reaction Research Role Side Source Structure System Triplet Multiple Birth abstracting amino group analog base cob(II)alamin cobamamide coenzyme B cofactor enzyme structure enzyme substrate complex epimerization ethanolamine hydroxyl group inhibitor/antagonist member methyl group research study suicide inactivation

项目摘要

The objectives of the project are to gain a better understanding of the 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 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.

该项目的目标是更好地了解基于自由基的机制酶促催化和腺基自由基在这些反应的启动中的作用。自由基反应在生物学中至关重要。虽然在生活系统是有害的，大自然使用自由基来在生物合成中执行困难的步骤和新陈代谢。腺苷泡剂（ADOCBL）依赖性酶代表酶的一个著名酶进行自由基介导的反应。乙醇胺氨 - 叶酶是一种依赖ADOCBL 来自细菌的酶。酶催化乙醇胺向乙醛和氨和包含该酶基因的细菌能够在乙醇胺上生长从磷脂的分解中，作为其唯一的碳，氮和能量的来源。酶是一组依赖ADOCBL的酶的成员，该酶催化了A的位置的互换氢原子和相邻碳原子上的取代基。这些酶使用Adocbl启动通过对Adocbl的钴 - 碳键的均化，从根本介导的重排以给予 COB（LL）丙酸和反应性5'-脱氧腺苷自由基。一些自由基中间体底物的反应达到浓度，以便可以通过电子顺磁观察到它们共振（EPR）光谱法。这些中间体将通过其特征来识别 EPR光谱中的核超细分裂模式。底物类似物导致自杀自由基的酶从正常反应途径中散发出来方法和通过鉴定反应产物。导致大于万亿倍的因素在酶结合的ADOCBL中的钴 - 碳键裂解速率的加速度将由探测使用核磁共振来测量手性5'-deutero形式的ADOCBL的表达式方法。催化周期中重排步骤的机制将通过测量进行探测使用同位素比质谱法对底物的氮动力学同位素效应的效果。基因来自不同种类细菌的酶将被克隆并在大肠杆菌中表达，蛋白质和蛋白质进行筛选以进行结晶。