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. ***
