MECHANISMS OF ENZYMATIC CATALYSIS

这项工作的长期目标是确定 酶催化化学反应的机制，以及 最终将这种机制中的各个步骤与 酶本身的特定官能团相互作用。 到 为此，我们使用了广泛的物理，化学， 动力学方法，以获得详细的了解伟大的 谷氨酸盐与谷氨酸盐之间形成的各种稳定的复合物 脱氢酶及其各种辅酶、底物和 效应器，最近研究了化学事件 参与这些复合体及其相互转化的 反应路径本身的发生顺序。 本提案中 我们从这种基础广泛的方法转向高度集中的 对该机制的一部分进行攻击，迄今为止， 最难以用常规方法分析的是 中心三元复合物的相互转化，一系列的五个 涉及这种氧化的大部分化学反应的步骤 脱氨反应 我们现在要集中精力 问题的两个方面： 1. 我们将确定发布的详细时间过程， 在过渡状态下吸收特定基团的H+离子 反应 在这些研究过程中，我们将继续 发展一种新的方法来解决瞬态问题 动力学现象 由于这种方法（时间进程比 方法）似乎是一个潜在的强大的新工具， 适用于酶的机制一般，我们认为它的 发展本身就是这个项目的目标。 2. 在我们研究的化学步骤中， 羰基转化为亚胺，我们将利用 我们最近发现羰基-O 18交换是一种直接的 亚胺形成的指标，提出四个具体问题：a. NADPH如何诱导亚胺形成？ B. 有哪些 五种或更多种瞬态的相对稳态浓度 在这个反应中，中间体？ C. 是否存在NH3结合位点或 NH3直接攻击羰基吗？ D. 有哪些 参与这些过程的功能组，以及如何 步骤本身与H+时间过程有关？ 谷氨酸盐的高分辨晶体结构 脱氢酶的研究是由一个小组进行的， 建立了密切的合作关系。 因此， 本提案中所述的工作旨在提供一种机制， 良好分离、化学定义明确且顺序正确的步骤， 适合即将到来的详细蛋白质结构。

The long range objective of this work is to determine the mechanisms by which enzymes catalyze chemical reactions, and ultimately to relate the individual steps in such mechanism to specific functional group interactions of the enzyme itself. To this end we have used a broad array of physical, chemical, and kinetic approaches to gain a detailed understanding of the great variety of stable complexes formed between glutamate dehydrogenase and its various coenzymes, substrates, and effectors, and more recently have studied the chemical events involved in the interconversions of those complexes and their order of occurrence on the reaction path itself. In this proposal we turn from this broad based approach to a highly focussed attack on the portion of the mechanism which has thus far been most resistant to analysis by conventional means--the interconversions of the central ternary complexes, a series of five steps which involve most of the chemistry of this oxidative deamination reaction. We will now concentrate our efforts on two aspects of the problem: 1. We will establish the detailed time course of the release and uptake of H+ ions of specific groups in the transient state of the reaction. In the course of these studies we will continue to develop a new approach to the resolution of transient-state kinetic phenomena. Since this approach (the time-course ratio method) appears to be a potentially powerful new tool which may be applicable to enzyme mechanisms in general, we consider its development to be a goal of this project in itself. 2. In our study of the chemistry of the steps involved in the conversion of the carbonyl group into an imine, we will make use of our recent finding that carbonyl-O18 exchange is a direct indicator of imine formation, asking four specific questions: a. How does NADPH induce imine formation? b. What are the relative steady-state concentrations of the five or more transient intermediate in this reaction? c. Is there an NH3 binding site or does NH3 attack the carbonyl group directly? d. What are the functional groups involved in these processes, and how are the steps themselves related to H+ time course? A high resolution crystallographic structure of glutamate dehydrogenase is now being carried out by a group with whom we have established a close collaboration. It is therefore the goal of the work described in this proposal to provide a mechanism of well resolved, chemically defined, and properly ordered steps to fit to the forthcoming detailed protein architecture.