Investigation of Enzyme Catalysis: Mechanism at the Levels of Subunit Function and the Chemical Transformation

本项目探讨催化活性与酶的物理、化学和结构性质之间的关系。研究酶催化的四个方面：(1)催化反应底物中共价转化的弱化学键和强化学键相互作用的性质。(2)变构效应物对酶催化过程活化能的影响。(3)酶的构象变化与催化的相互关系。(4)代谢途径中催化顺序步骤的双酶复合物的代谢物转移机制。为了研究这些现象，我们选择了四种酶系统进行详细的机制研究：(1)人肝和马肝乙醇脱氢酶，(2)大肠杆菌中需要PLP的色氨酸合成酶双酶，(3)大肠杆菌中需要胱氨酸- γ合成酶的PLP，以及(4)羊肝中的醛脱氢酶。该项目强调使用快速动力学技术（特别是快速扫描和单波长停流紫外可见光谱）来研究这些系统。其他对项目重要的技术包括停流荧光光谱1H和31P FT NMR，酶晶体的紫外可见显微分光光度法和酶侧链残基的化学修饰。这些技术将用于检测和识别催化中的化学中间体，寻找与催化有关的构象变化，并检查变构效应剂对催化路径上化学中间体相互转化的影响。这些研究有望显著推进我们对酶催化的一些最不为人所知的方面的理解。

This project explores the relationship betweeen the catalytic activities and the physical, chemical, and structural properties of enzymes. Four aspects of enzyme catalysis will be studied: (1) The nature of the weak and strong chemical bonding interactions which catalyze covalent transformations in the reacting substrate. (2) The influence of allosteric effectors on the activation energies of enzyme-catalyzed processes. (3) The interrelation between enzyme conformational change and catalysis. (4) Metabolite transfer mechanistic in bienzyme complexes which catalyze sequential steps in a metabolic pathway. To investigate these phenomena, four enzyme systems are selected for detailed mechanistic studies: (1) the liver alcohol dehydrogenases from human and horse liver, (2) the bienzyme, PLP-requiring tryptophan synthase from E. coli, (3) the PLP requiring cystathionine gamma-synthase from E. coli, and (4) aldehyde dehydrogenase from sheep liver. The project emphasizes the use of rapid kinetic techniques (especially rapid scanning and single wavelength stopped-flow UV-visible spectroscopy) to study these systems. Other techniques of importance to the project include stopped-flow fluorescence spectroscopy 1H and 31P FT NMR, UV-visible microspectrophotometry of enzyme crystals and chemical modification of enzyme side chain residues. These techniques will be used to detect and identify chemical intermediates in catalysis, to search for conformational changes linked to catalysis, and to examine the influence of allosteric effectors on the interconversion of chemical intermediates on the catalytic path. These studies are expected to significantly advance our understanding of some of the least well understood aspects of enzyme catalysis.