Mechanisms of Enzymatic Catalysis

9513398费舍尔这个实验室的长期目标是了解酶作为起作用的生物机器。我们希望了解它们的机制的化学，它们的结构特征如何启动和控制该化学，以及它们操作的能量学如何与它们在哺乳动物谷氨酸脱氢酶催化反应的反应时间过程中发生的化学和结构事件有关；2.将该反应的时间过程分解为一系列精确定义的“开放”和“封闭”阶段。本研究主要针对牛肝谷氨酸脱氢酶的化学机制进行了研究。在我们以前的工作中，我们已经识别和表征了涉及的大多数重要的酶复合体，并确定了其中许多酶复合体在反应时间过程中的位置。我们最近的进展导致了一种新的预氢化物转移电荷转移络合物的发现；初步鉴定了最终所需的氢化物后络合物(乙醇胺形式)；发现在哺乳动物酶中氢化物转移之前发生的必须的质子释放步骤被转移到相应细菌酶形式的较晚阶段，其化学和活性部位结构似乎在其他方面是相同的。我们还开发了一些新的研究工具，包括一种新的暂态动力学同位素效应理论(我们已经在实验中演示了它的应用)。我们现在建议应用这些方法和一些新的实验方法来解决两个特定的目标。这些新方法包括使用一种非常简单的基于质量定律的动力学方法，将我们现在可以用最近获得的仪器产生的组分浓度时间过程整合到一个完全定义的方案中。我们还将应用并继续发展我们的新的暂态动力学同位素效应理论。第二个目标将通过各种多混合“动力学跳跃”实验来实现，以确定中间络合物的构象状态。分子工程，即改变蛋白质以满足人类需求的能力，是一个明确的国家目标。满足这一需求所需的信息是在详细水平上了解一系列结构-功能关系的形式。目前该领域的方法是结合蛋白质晶体结构信息和这些结构的定点突变。然而，如果对这些生物机器的作用机制没有更详细的了解，我们就不能提出适当的问题。这里提出的工作就是为了提供这样的知识。这个实验室的长期目标是了解酶作为起作用的生物机器。我们希望了解它们的机制的化学，它们的结构特征是如何启动和控制这种化学的，以及它们的操作的能量学是如何与它们的反应序列中发生的化学和结构事件有关的。我们的近期目标是：1.完成哺乳动物谷氨酸脱氢酶催化反应的反应时间进程的动力学解析；以及2，将该反应的时间进程分解为一系列精确定义的“开放”和“封闭”相。本研究主要针对牛肝谷氨酸脱氢酶的化学机制进行了研究。我们开发了两个重要的新工具，使我们能够在更深的水平上理解酶催化的机理：1.一个非常简单的基于质量定律的动力学方法，将我们现在可以用最近获得的装置产生的组分-浓度时间过程整合到一个完全定义的方案中；2，一个新的瞬时动力学同位素效应理论。分子工程，即改变蛋白质以满足人类需求的能力，是一个明确的国家目标。这里提出的工作就是为了提供这样的知识。***

9513398 Fisher The long term goal of this laboratory is to understand enzymes as functioning biological machines. We hope to learn the chemistry of their mechanisms, how their structural features initiate and control that chemistry, and how the energetics of their operation is related to the chemical and structural events that occur in their reaction time course of the mammalian glutamate dehydrogenase catalyzed reaction; and 2. To resolve the time course of that reaction into a series precisely defined "open" and "closed" phases. This proposal is focused on the chemical mechanism of bovine liver glutamate dehydrogenase. In our previous work we have identified and characterized most of the important enzyme complexes involved and have established the locations of many of them in the reaction time course. Our recent progress has resulted in the discovery of a new prehydride transfer charge-transfer complex; the preliminary identification of the final required post hydride complex (a carbinolamine form); the finding that an obligatory proton release step which occurs prior to hydride transfer in the mammalian enzyme is shifted to a much later phase in a corresponding bacterial enzyme form whose chemistry and active-site architecture appear to be otherwise identical. We have also developed some new investigative tools, including a new theory of transient state kinetic isotope effects (whose application we have demonstrated experimentally). We propose now to apply these methods and some new experimental approaches to solve two specific objectives. These new methods include the use of a very simple mass-law based kinetic approach to integrate the component-concentration time courses we can now produce using a recently acquired apparatus into a completely defined scheme. We w ill also apply and continue to develop our new transient-state kinetic isotope effect theory. The second objective will be pursued using a variety of multimixing "kinetic-jump" experiments to determine the co nformational state of the intermediate complexes. Molecular engineering, the ability to alter proteins to meet man's needs is a well defined national 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 questions. The work proposed here is intended to provide just such knowledge. %%% The long term goal of this laboratory is to understand enzymes as functioning biological machines. We hope to learn the chemistry of their mechanisms, how their structural features initiate and control that chemistry, and how the energetics of their operations is related to the chemical and structural events that occur in their reaction sequences. Our immediate objectives are: 1. To complete the kinetic resolution of the reaction time course of the mammalian glutamate dehydrogenase catalyzed reaction; and 2, To resolve the time course of that reaction into a series of precisely defined "open" an "closed" phases. This proposal is focused on the chemical mechanism of bovine liver glutamate dehydrogenase. We have developed two important new tool which we will apply to enable us to understand the mechanism of enzymatic catalysis at a deeper level: 1. a very simple mass-law based kinetic approach to integrate the component-concentration time courses we can now produce using a recently acquired apparatus into a completely defined scheme; and 2, a new transient-state kinetic isotope effect theory. Molecular engineering, the ability to alter proteins to meet man's needs, is a well defined national goal. The work proposed here is intended to provide just such knowledge. ***