Our long range goal is to extend our knowledge of the basic nature of enzymatic catalysis to a more fundamental level than currently available experimental approaches permit. A major component of detailed enzyme mechanisms as customarily written is a sequential shuffling of protons hydrogen bonded to varying functional groups of the enzyme and its reactant species as the reaction proceeds through its various steps. Such proton shifts are usually inferred from arguments based on kinetic and structural information combined with chemical intuition.
The aims of this proposal are to develop and to begin to apply a new approach in which the movement of specific protons in previously identified individual reaction steps can be detected directly. This approach is based on the combining of two different developments from the recent work of this laboratory: 1. Our demonstrated ability to produce transient-state time courses of individual complexes in an enzyme reaction; 2., the development and experimental verification of a novel rigorous theory of the time-dependent behavior of transient-state KIE's which yields valid intrinsic KIE's for each reaction step. This method produces results which differ substantially from those currently in use. We will develop and test this new approach on the well studied reaction of bovine liver L-glutamate dehydrogenase (blGDH). The basic method employed is the multiwavelength transient-state kinetic measurement of the reaction in both H2O and D2O solvents. Such studies will be carried out on the following kinds of systems: 1. the full reaction time course under various conditions; 2. several known partial reactionsL and 3. some signals which are specific to an individual bond-making or bond-breaking event. A detailed reaction mechanism based on crystallographic and computer modeled structures will serve as the hypothesis to be tested by our results. The validity of our interpretations will in turn be tested by application of our transient-state solvent KIE approach to a number of relevant non-enzymatic reactions whose detailed mechanisms are well established.
