Role of NADPH in the regulation of NADP-specific isocitrate dehydrogenase from pig heart.

The present results show that the NADP specific isocitrate dehydrogenase from pig heart exhibits a time lag before the reaction rate approaches a constant value at low metal ion concentrations. Addition of NADPH or EDTA to the assay mixture abolished the lag, and will under certain conditions activate the enzyme.The lag time increased with increasing concentrations of isocitrate and decreased with increasing enzyme concentration. The NADP and metal ion concentration affected the lag in a complex manner. At low NADP and isocitrate concentration, the lag was reduced 50% by an NADPH concentration of less than 2 μm. Stopped flow experiments showed that premixing of NADP or NADPH with the enzyme abolished the effect of NADPH on the lag time. NADPH activated the enzyme at high NADP concentrations. This activating effect could be accounted for by removal of substrate inhibition by NADP.Evidence was obtained to show that the effect of NADPH on the activity was caused by binding of the reduced coenzyme to a site separate from the normal coenzyme binding site. Binding of metal ions by the reduced coenzyme is probably of importance as EDTA affects the lag time and activity in a manner similar to NADPH. The NADPH effect seems to be a general property of NADP-linked isocitrate dehydrogenases.