Mechanisms of catalysis by an alpha-amino acid dehydrogenase

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的反应涉及比通常设想的多得多的步骤，所涉及的许多复合物中的每一个都占据了许多容易相互转化的构象状态，因此，给定酶反应的反应最好以多维构象对反应步骤表面上的多个迹线的形式表示。作为一个假设，我们提供了一个新的扩展机制的L-苯丙氨酸脱氢酶的反应，其中包括步骤和复合物的发生是公认的和几个复合物和步骤的发生尚未建立似乎是实验测试。我们的具体目标是基于我们的酶作为分子机器的观点。因此，我们探讨它的机制，它的结构和能量的运作。因此，目标1涉及在各种条件下的化学反应时间过程的详细调查，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它们的组分。目的2是建立相应的构象的时间过程（或课程）的这个反应，并与观察到的差异，结构和热力学性质，在其个别步骤，使用两个建立量热方法和一个新开发的中间复杂的平衡方法。目的3是扩展我们的知识的活性位点区域的配合物的原子结构还没有这样的特点，使用合作的X射线晶体学研究。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是为了推进我们对这个问题的理解，但更重要的是，探索新的方法来这个领域。

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.