The Mechanism of the Bovine Liver Glutamate Dehydrogenase Catalayzed Reaction

这个实验室的长期目标是了解酶催化的详细的化学、热力学和结构基础，并了解蛋白质分子作为生物机器的一般功能。这项工作的重点是详细研究在牛肉肝脏谷氨酸脱氢酶(BlGDH)催化反应中发生的化学事件和蛋白质构象的变化。这些研究涉及应用瞬时(实时)动力学方法来实现两个特定的目标：1.完成对blGDH反应的反应时间历程的动力学解析。虽然研究人员在上一个授权期的工作是针对反应中心部分的一系列四个络合物的动力学能力的表征和证明，但他现在将把注意力转向较少了解的较早步骤2。将blGDH反应的时间过程分解为一系列精确定义的“开放”和“关闭”阶段。对细菌形式的酶(CsGDH)的晶体结构研究表明，该酶既存在一种开放形式，也存在至少两种不同的封闭形式。研究人员已经获得了在酶催化反应中发生了几个这样的活性部位裂隙打开和关闭步骤的物理和动力学证据。他将继续努力在正向和反向反应中确定这些阶段在可用pH范围内的发生点、持续时间和演替。分子工程是一个重要的目标，即改变蛋白质以满足人类需求的能力。满足这一需求所需的信息是在详细水平上了解一系列结构-功能关系的形式。目前该领域的方法是结合蛋白质晶体结构信息和这些结构的定点突变。然而，如果对这些生物机器的作用机制没有更详细的了解，我们就不能为这种方法制定适当的战略。这项研究将提供这样的知识。

9816697The long-range goal of this laboratory is to learn the detailed chemical, thermodynamic and structural basis of enzymatic catalysis in particular and to understand how protein molecules function as biological machines in general. The work proposed here is focused on a detailed examination of the chemical events and changes in protein conformation that occur in the reaction catalyzed by beef liver glutamate dehydrogenase (blGDH). These studies involve the application of transient-state (real-time) kinetic approaches to two specific aims:1. To complete the kinetic resolution of the reaction time course of the blGDH reaction. While the work of the investigator in the previous grant period was directed at the characterization and proof of kinetic competence of a sequence of four complexes in the central portion of the reaction, he will now turn his attention to the less understood earlier steps.2. To resolve the time course of the blGDH reaction into a series of precisely defined "open" and "closed" phases. Crystal structure studies of the bacterial form of the enzyme (csGDH) have demonstrated the existence of both an open and at least two different closed forms of the enzyme. The investigator has obtained both physical and kinetic evidence of the occurrence of several such active-site cleft opening and closing steps during the enzyme-catalyzed reaction. He will continue his ongoing efforts to identify the points of occurrence, duration, and succession of these phases over the usable pH range in both the forward and the reverse reactions.Molecular engineering, the ability to alter proteins to meet humanitys needs, is an important goal. The information required to fulfill this need is in the form of understanding a series of structure-function relationships at a detailed level. The current approach in the field is the combination of protein crystal structure information and site directed mutagenesis of those structures. However, without a more detailed knowledge of the mechanisms of action of these biological machines, we cannot formulate the proper strategy for such an approach. This research will provide just such knowledge.