The long range objective of this work is to determine the mechanisms by which enzymes catalyze chemical reactions, and ultimately to relate the individual steps in such mechanism to specific functional group interactions of the enzyme itself. To this end we have used a broad array of physical, chemical, and kinetic approaches to gain a detailed understanding of the great variety of stable complexes formed between glutamate dehydrogenase and its various coenzymes, substrates, and effectors, and more recently have studied the chemical events involved in the interconversions of those complexes and their order of occurrence on the reaction path itself. In this proposal we turn from this broad based approach to a highly focussed attack on the portion of the mechanism which has thus far been most resistant to analysis by conventional means--the interconversions of the central ternary complexes, a series of five steps which involve most of the chemistry of this oxidative deamination reaction. We will now concentrate our efforts on two aspects of the problem: 1. We will establish the detailed time course of the release and uptake of H+ ions of specific groups in the transient state of the reaction. In the course of these studies we will continue to develop a new approach to the resolution of transient-state kinetic phenomena. Since this approach (the time-course ratio method) appears to be a potentially powerful new tool which may be applicable to enzyme mechanisms in general, we consider its development to be a goal of this project in itself. 2. In our study of the chemistry of the steps involved in the conversion of the carbonyl group into an imine, we will make use of our recent finding that carbonyl-O18 exchange is a direct indicator of imine formation, asking four specific questions: a. How does NADPH induce imine formation? b. What are the relative steady-state concentrations of the five or more transient intermediate in this reaction? c. Is there an NH3 binding site or does NH3 attack the carbonyl group directly? d. What are the functional groups involved in these processes, and how are the steps themselves related to H+ time course? A high resolution crystallographic structure of glutamate dehydrogenase is now being carried out by a group with whom we have established a close collaboration. It is therefore the goal of the work described in this proposal to provide a mechanism of well resolved, chemically defined, and properly ordered steps to fit to the forthcoming detailed protein architecture.
