The Mechanism of the Bovine Liver Glutamate Dehydrogenase Catalayzed Reaction

9816697该实验室的长期目标是学习酶催化的详细化学，热力学和结构基础，特别是了解蛋白质分子如何作为生物机器发挥作用。这里提出的工作是集中在一个详细的检查的化学事件和蛋白质构象的变化，发生在牛肝谷氨酸脱氢酶（blGDH）催化的反应。这些研究涉及应用瞬态（实时）动力学的方法，以两个特定的目标：1。完成blGDH反应的反应时间进程的动力学解析。虽然研究人员在上一个资助期的工作是针对反应中心部分的一系列四个复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不了解的早期步骤。将blGDH反应的时间过程分解为一系列精确定义的“开放”和“闭合”阶段。细菌形式的酶（csGDH）的晶体结构研究表明，存在开放和至少两种不同的封闭形式的酶。研究者已经获得了在酶催化反应期间发生几个这样的活性位点裂缝打开和关闭步骤的物理和动力学证据。他将继续他正在进行的努力，以确定发生点，持续时间，并在可用的pH值范围内，在正向和反向reaction.Molecular工程，改变蛋白质，以满足人类的需要的能力，这些阶段的继承是一个重要的目标。满足这一需求所需的信息是以详细理解一系列结构-功能关系的形式。该领域目前的方法是结合蛋白质晶体结构信息和这些结构的定点诱变。然而，如果不对这些生物机器的作用机制有更详细的了解，我们就无法为这种方法制定适当的策略。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.