Stabilization of noncovalent intermediates in enzymatically catalyzed reactions

Reactive intermediate enzyme complexes are difficult to study directly and the use of physical methods requiring observation periods of more than a second has not been possible heretofore. Here we introduce a simple approach, the "Le Chatelier forcing method" which does for the first time produce significant concentrations of such kinetically competent central intermediates observable for extended periods of time. The method involves only the forcing of the accumulation of intermediate complexes at thermodynamic equilibrium by the use of high reactant concentrations working against a high concentration of a product, combined with a valid and applicable method of analysis. We demonstrate this approach using the glutamate dehydrogenase catalyzed reaction with the reaction product ammonia as a "dam" to oppose the forward driving force of NADP and l-glutamate. We demonstrate the accumulation of substantial amounts measurable amounts of stable enzyme-NADPH-alpha-carbinolamine and alpha-iminoglutarate complexes in three different alpha-amino acid dehydrogenases. We describe the manipulation of such Le Chatelier forced equilibria to increase the prominence of particular species and discuss the implications of these findings for previously unattainable experimental approaches.