On the mechanism of NADP+-linked isocitrate dehydrogenase from heart mitochondria. I. The kinetics of dissociation of NADPH from its enzyme complex

The kinetics of dissociation of NADPH from its complex with isocitrate dehydrogenase, and from the abortive complex of enzyme, Mg2+, isocitrate and NADPH, have been studied in phosphate and triethanolamine buffers by means of rapid fluorescence measurements. The reactions are complex, and it is suggested that a conformational equilibrium of each of the complexes is involved, and that this conformational change is also responsible for a slow approach to the steady-state rate of oxidative decarboxylation observed previously in triethanolamine buffer under certain conditions (K. Dalziel, N. McFerran, B. Matthews & C.H. Reynolds, Biochem. J. 171, 743-750 (1978) ). It is concluded that release of free NADPH product is not the rate-limiting step in oxidative decarboxylation in the steady state. The validity of the ligand displacement method used to measure the dissociation kinetics of the enzyme-NADPH complex has been studied by computer simulation.