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：
8120800
负责人：
Harvey F. Fisher
金额：
$ 27.66万
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：
2007
资助国家：
美国
起止时间：
2007-09-07 至 2012-07-31
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）：我们研究计划的远距离目标是确定酶促催化的基本基础。我们目前策略的基本原理是我们对酶催化反应的证据的越来越多的认识涉及的步骤要比一般设想的要多得多，即所涉及的许多复合物中的每一个都占据了许多易于互动的构象状态，因此，在多个示波器上，在多个有害的反应中最好地表达了在多个有限度上的反应。作为一个假设，我们为L-苯丙氨酸脱氢酶反应提供了新扩展的机制，其中包括步骤和复合物，其发生良好的发生以及尚未确定的几个复合物和步骤似乎是可以在实验上测试的。我们的具体目的是基于我们将酶作为分子机器的看法。因此，我们探索了其机制，其结构和运作的能量。因此，AIM 1涉及对各种条件下化学反应时间过程的详细研究，使用我们实验室中开发的各种瞬时状态动力学解决了分量的总反应步骤。 AIM 2是建立该反应的相应构象时过程（或过程），并使用已建立的量热法和新开发的中间复杂的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特性相关联。 AIM 3是扩展我们对使用协作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。酶的活性在于每个生命过程。然而，我们目前对其机制的了解占其催化能力的十分之一。这里提出的实验不仅旨在提高我们对这个问题的理解，而且更重要的是，探索了该领域的新方法。