Mechanisms of catalysis by an alpha-amino acid dehydrogenase

α-氨基酸脱氢酶的催化机制

基本信息

批准号：
8051920
负责人：
Harvey F. Fisher
金额：
$ 14.4万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-15 至 2011-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long range goal of our research program is to determine the fundamental basis of enzymatic catalysis. The rationale underlying our current strategies is our growing awareness of the evidence that enzyme- catalyzed reactions involve substantially more steps than are generally envisioned, that each of the many complexes involved occupy a number of readily interconvertible conformational states, and that, as a result, the reaction of a given enzyme reaction is best expressed in the form of multiple traces on a multi- dimensional conformation vs. reaction step surface. As a hypothesis we offer a newly extended mechanism for the L-phenylalanine dehydrogenase reaction which includes both steps and complexes whose occurrence is well established and several complexes and steps whose occurrence has not yet been established which appear to be experimentally testable. Our Specific Aims are based on our view of an enzyme as a molecular machine. As such we explore its mechanism, its structure and the energetics of its operation. Aim 1, therefore, involves the detailed investigation of the chemical reaction time course under various conditions, resolving gross reaction steps into their components using a variety of transient state kinetics developed in our laboratory. Aim 2 is to establish the corresponding conformational time course (or courses) of this reaction, and to relate the differences observed to both structural and thermodynamic properties in its individual steps using both established calorimetric approaches and a newly developed intermediate complex-at-equilibrium approach. Aim 3 is to extend our knowledge of the atomic structure of the active site regions of complexes not yet so characterized using collaborative X-ray crystallographic studies. The activity of enzymes lies at the basis of every life process. Yet, our current knowledge of their mechanisms accounts for less than one ten-thousandth of their catalytic power. The experiments proposed here are intended not only to advance our understanding of this problem but, more importantly, to explore new approaches to this field.

描述（由申请人提供）：我们研究计划的长期目标是确定酶催化的基本基础。我们当前策略的基本原理是我们越来越意识到有证据表明酶催化反应涉及的步骤比通常想象的要多得多，所涉及的许多复合物中的每一个都占据了许多易于相互转换的构象状态，因此，给定酶反应的反应最好以多维构象与反应步骤表面上的多条迹线的形式表达。作为一种假设，我们为 L-苯丙氨酸脱氢酶反应提供了一种新扩展的机制，其中包括其发生已被充分确定的步骤和复合物，以及其发生尚未确定但似乎可以通过实验测试的几个复合物和步骤。我们的具体目标基于我们将酶视为分子机器的观点。因此，我们探索其机制、结构及其运行的能量学。因此，目标 1 涉及对各种条件下化学反应时间过程的详细研究，使用我们实验室开发的各种瞬态动力学将总反应步骤分解为其组成部分。目标 2 是建立该反应相应的构象时间过程（或多个过程），并使用已建立的量热方法和新开发的中间复合物平衡方法将观察到的差异与其各个步骤中的结构和热力学性质联系起来。目标 3 是扩展我们对尚未使用协作 X 射线晶体学研究表征的复合物活性位点区域的原子结构的了解。酶的活性是每个生命过程的基础。然而，我们目前对其机制的了解还不到其催化能力的万分之一。这里提出的实验不仅旨在增进我们对这个问题的理解，更重要的是探索该领域的新方法。