Mechanisms of catalysis by an alpha-amino acid dehydrogenase

描述(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。我们目前策略的基本原理是我们越来越意识到有证据表明，酶催化的反应涉及的步骤比通常想象的要多得多，所涉及的许多络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，给定的酶反应的反应最好地以多维构象与反应步骤表面上的多个痕迹的形式表达。作为一个假设，我们提出了一个新的扩展的L-苯丙氨酸脱氢酶反应机理，它包括步骤和络合物，它们的存在已得到很好的证实，以及几个尚未确定其发生的络合物和步骤，似乎是可实验检验的。我们的具体目标是基于我们对酶作为分子机器的看法。因此，我们探讨了它的机制、结构和运行的能量学。因此，目标1涉及详细研究不同条件下的化学反应时间过程，使用我们实验室开发的各种暂态动力学将总反应步骤分解为它们的组成部分。目标2是建立该反应的相应构象时间进程，并使用已建立的量热方法和新开发的中间络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是扩展我们对尚未用协作X射线结晶学研究表征的络合物的活性中心区域的原子结构的知识。酶的活性是每个生命过程的基础。然而，我们目前对它们的机理的了解还不到它们催化能力的万分之一。这里提出的实验不仅是为了促进我们对这个问题的理解，更重要的是，探索这一领域的新方法。

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.