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

这个项目探讨了催化剂和催化剂之间的关系。 活动和物理，化学和结构 酶的性质。 酶催化的四个方面 研究：（1）弱化学和强化学的性质 催化共价转化的键合相互作用 在反应基质中。 (2)变构的影响 酶催化反应活化能的影响因素 流程. (3)酶之间的相互关系 构象变化和催化作用。 (4)代谢物转移 双酶复合物的催化机理 代谢途径中的步骤。 调查这些 现象，选择了四种酶系统进行详细的 机制研究：（1）肝脏乙醇脱氢酶 从人类和马的肝脏，（2）双酶，PLP需要 色氨酸合成酶;大肠杆菌，（3）PLP需要 来自E.（4）醛 绵羊肝脏脱氢酶。 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 UV-可见光谱）来研究这些系统。 其他 该项目的重要技术包括停流 荧光光谱1H和31 P FT NMR，UV-可见 酶晶体显微分光光度法 酶侧链残基的修饰。 这些技术 将用于检测和识别化学中间体， 催化，以寻找与 催化，并检查变构的影响， 化学中间体相互转化的影响因素 催化途径。这些研究有望 大大地推进了我们对一些最不重要的 酶催化的一些方面。

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.