Mechanisms of Enzymatic Catalysis

9513398 Fisher本实验室的长期目标是了解酶作为功能生物机器。 我们希望了解它们的化学机制，它们的结构特征如何启动和控制该化学，以及它们的操作的能量学如何与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相关;和2. 将反应的时间进程分解为一系列精确定义的“开放”和“封闭”阶段。 本论文主要研究牛肝谷氨酸脱氢酶的化学机制。 在我们以前的工作中，我们已经确定和表征了大多数重要的酶复合物，并建立了它们中的许多在反应时间过程中的位置。 我们最近的进展是发现了一种新的预氢化物转移电荷转移络合物，初步鉴定了最终所需的后氢化物络合物，（甲醇胺形式）;发现在哺乳动物酶中发生在氢化物转移之前的强制性质子释放步骤在相应的细菌酶形式中转移到更晚的阶段，站点体系结构在其他方面似乎是相同。 我们还开发了一些新的研究工具，包括瞬态动力学同位素效应的新理论（我们已经通过实验证明了其应用）。 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具体的目标。 这些新的方法包括使用一个非常简单的质量定律为基础的动力学方法来整合的成分浓度的时间过程中，我们现在可以使用最近获得的设备到一个完全定义的计划。 我们还将应用并继续发展我们新的瞬态动力学同位素效应理论。 第二个目标将追求使用各种多重混合的“动力学跳跃”实验，以确定中间复合物的构象状态。 分子工程，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以详细理解一系列结构-功能关系的形式。 该领域目前的方法是结合蛋白质晶体结构信息和这些结构的定点诱变。 然而，如果不对这些生物机器的作用机制有更详细的了解，我们就无法提出适当的问题。 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功能生物机器。 我们希望了解它们的化学机制，它们的结构特征如何启动和控制化学，以及它们的操作能量如何与它们的反应序列中发生的化学和结构事件相关。 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时间过程的动力学解析; 2.将该反应的时间过程解析为一系列精确定义的“开放”和“封闭”阶段。 本论文主要研究牛肝谷氨酸脱氢酶的化学机制。 我们已经开发了两个重要的新工具，我们将应用它们使我们能够在更深层次上理解酶催化的机制：1。一个非常简单的质量定律为基础的动力学方法，以整合的成分浓度的时间课程，我们现在可以使用最近获得的设备到一个完全定义的计划;和2，一个新的瞬态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要的能力，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. ***