The long-range goal of this laboratory is to learn the detailed chemical, thermodynamic and structural basis of enzymatic catalysis in particular and to understand how protein molecules function as biological machines in general. The work proposed here is focused on a detailed examination of the chemical events and changes in protein conformation that occur in the reaction catalyzed by beef liver glutamate dehydrogenase (blGDH). These studies involve the application of transient-state (real-time) kinetic approaches to two specific aims:
1. To complete the kinetic resolution of the reaction time course of the blGDH reaction. While the work of the investigator in the previous grant period was directed at the characterization and proof of kinetic competence of a sequence of four complexes in the central portion of the reaction, he will now turn his attention to the less understood earlier steps.
2. To resolve the time course of the blGDH reaction into a series of precisely defined "open" and "closed" phases. Crystal structure studies of the bacterial form of the enzyme (csGDH) have demonstrated the existence of both an open and at least two different closed forms of the enzyme. The investigator has obtained both physical and kinetic evidence of the occurrence of several such active-site cleft opening and closing steps during the enzyme-catalyzed reaction. He will continue his ongoing efforts to identify the points of occurrence, duration, and succession of these phases over the usable pH range in both the forward and the reverse reactions.
Molecular engineering, the ability to alter proteins to meet humanitys needs, is an important 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 strategy for such an approach. This research will provide just such knowledge.
