A kinetic mechanism of the allosteric control of enzyme-coenzyme binding: glutamate dehydrogenase-NADPH-phosphate-acetate-hydrogen ion interactions.

We have previously characterized the thermodynamic relationships which govern the dissociation of NADPH from bovine liver glutamate dehydrogenase and the allosteric control of that mechanically and physiologically important process by a variety of effectors. We have found that the cooperative occupancy of a specific anion binding, while the occupancy of a second allosteric acetate binding site disrupts that anion binding site and opposes those effects (Singh & Fisher, 1994). We report here the results of transient-state studies on the kinetics of the various processes involved in this complex equilibrium. We find that the only intrinsically slow steps are those of NADPH binding and dissociation, that the complex kinetic behavior of the overall system is due solely to very rapid equilibrium binding processes involving phosphate, acetate, and hydrogen ions, and that these ions exert their various effects on the kinetics of the binding process by altering the equilibrium concentrations of the two kinetically significant reactive species, E and E-NADPH. The slow intrinsic rates of NADPH association and dissociation are ascribed to a ligand-induced conformational change involving a major alteration in the degree of closure of the enzyme's active-site cleft.