The long range goal of our research program is to determine the fundamental basis of enzymatic catalysis. The rationale underlying our current strategies is our growing awareness of the evidence that enzyme- catalyzed reactions involve substantially more steps than are generally envisioned, that each of the many complexes involved occupy a number of readily interconvertible conformational states, and that, as a result, the reaction of a given enzyme reaction is best expressed in the form of multiple traces on a multi- dimensional conformation vs. reaction step surface. As a hypothesis we offer a newly extended mechanism for the L-phenylalanine dehydrogenase reaction which includes both steps and complexes whose occurrence is well established and several complexes and steps whose occurrence has not yet been established which appear to be experimentally testable.
Our Specific Aims are based on our view of an enzyme as a molecular machine. As such we explore its mechanism, its structure and the energetics of its operation.
Aim 1, therefore, involves the detailed investigation of the chemical reaction time course under various conditions, resolving gross reaction steps into their components using a variety of transient state kinetics developed in our laboratory.
Aim 2 is to establish the corresponding conformational time course (or courses) of this reaction, and to relate the differences observed to both structural and thermodynamic properties in its individual steps using both established calorimetric approaches and a newly developed intermediate complex-at-equilibrium approach.
Aim 3 is to extend our knowledge of the atomic structure of the active site regions of complexes not yet so characterized using collaborative X-ray crystallographic studies. The activity of enzymes lies at the basis of every life process. Yet, our current knowledge of their mechanisms accounts for less than one ten-thousandth of their catalytic power. The experiments proposed here are intended not only to advance our understanding of this problem but, more importantly, to explore new approaches to this field.
|Fisher, Harvey F; Maniscalco, Steven J; Tally, Jon et al. (2009) Application of the second rule of transient-state kinetic isotope effects to an enzymatic mechanism. Biochemistry 48:12265-71|