Use of a Bisubstrate Inhibitor to Distinguish Between Isocitrate Dehydrogenase Isozymes

Abstract

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 addncts. 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.

Key Words: :

• Isocitrate dehydrogenase
• Oxoglutarate
• NAD(P)H
• Inhibitor
• Coenzyme