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。至 为此，我们使用了广泛的物理、化学和 用动力学的方法来详细了解 谷氨酸之间形成的各种稳定的络合物 脱氢酶及其各种辅酶、底物和 效应器，以及最近研究的化学事件 参与了这些复合体和它们的相互转化 反应路径本身的发生顺序。在本建议书中 我们从这种基础广泛的方法转向高度集中的 对到目前为止一直存在的机制部分的攻击 最不愿用常规方法进行分析-- 中心三元络合物的相互转化，五个系列 涉及这种氧化的大部分化学成分的步骤 脱氨反应。我们现在将集中力量在以下方面 问题的两个方面： 1.我们将确定发布的详细时间进程，并 在瞬变状态下特定基团的H离子吸收 反应。在这些研究过程中，我们会继续 发展一种解决暂态问题的新方法 动力学现象。由于这种方法(时程比 方法)似乎是一个潜在的强大的新工具，它可能 一般适用于酶机制，我们认为其 发展本身就是这个项目的一个目标。 2.在我们对所涉及的步骤的化学研究中 将羰基转化为亚胺，我们将利用 我们最近的发现中，羰基-O18交换是一种直接的 亚胺形成的指示剂，提出四个具体问题：a. NADPH如何诱导亚胺的形成？B.有什么是 五种或五种以上暂态的相对稳态浓度 这一反应的中间体？C.是否存在NH3结合部位或 NH3是否直接攻击羰基？D.有什么是 这些过程中涉及的官能团，以及 步骤本身与H时间进程相关吗？ 谷氨酸的高分辨晶体结构 脱氢酶现在是由一个小组进行的，我们和这个小组一起 已经建立了密切的合作关系。因此，它的目标是 本提案中描述的提供一种机制的工作 良好的解析、化学定义和适当的有序步骤 适合即将到来的详细的蛋白质结构。

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.