Use of a bisubstrate inhibitor to distinguish between isocitrate dehydrogenase isozymes.

Bisubstrate inhibitors, obtained by covalently linking 2-oxoglutarate with NAD+ and NADP+, were synthesized and tested for their ability to inhibit NAD+- and NADP+-dependent isocitrate dehydrogenases from pig heart mitochondria. The NADP+-dependent enzyme was specifically inhibited by the NADP oxoglutarate adduct and not by the NAD adduct. The NADP adduct was competitive with both coenzyme and substrate, isocitrate. In contrast, the NAD+-dependent enzyme was inhibited by both adducts. NAD oxoglutarate is competitive with both NAD+ and isocitrate while the NADP adduct is competitive with isocitrate but not with NAD+. Nevertheless conditions could be set up so that use of these inhibitors would be feasible for a metabolic study.