STRUCTURE AND MECHANISMS OF ACTION OF ENZYMES

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The long-range objectives of this laboratory are to determine the mechanisms by which enzymes catalyze reactions, and in a more general way to determine the nature of biological interactions at the molecular level. To these we have now added an attempt to gain some understanding of the energetics of the catalytic process and to learn the relationship between the energetics of individual steps and the chemical events occurring in those steps. For some years we have focused our investigations on the glutamic dehydrogenase system which offers a wide variety of phenomena with which to pursue such studies. Having already blocked out the order of occurrence of the nine major complexes formed along the reaction time course, we now turn to the study of the detailed chemical events involved in the interconversions of those complexes. In this proposal we focus our attention on two critical areas of the reaction sequence: 1) What is the role of the carbonyl group of Alpha-ketoglutarate, and at precisely which point does the required water molecule enter the reaction course? This aim will be explored through the use of an analog lacking the carbonyl group and by a variety of 0 1 8 studies. 2) What are the events and complexes preceding and accompanying the hydride transfer step? This will be explored by a study of very pertinent model reactions and by parallel studies on the early transient steps. This latter study will include cryoenzymology and alternative kinetic approach to the study of transients which may have some application to the study of enzymes in general. Finally, we will attempt to relate a recently discovered temperature-dependent poised equilibrium between two isomeric forms of the free enzyme to changes in hydration and (or) to the large negative DeltaCp's observed in many of the complexes of pyridine nucleotide dehydrogenases. These initial experimental probes are a prelude to the definition of a serious effort in this area on our part to determine whether, as Jencks has suggested, part of the substrate binding energy can be channeled into driving a later catalytic step, and if so, how it is accomplished.