The β and γ subunits play distinct functional roles in the αβγ heterotetramer of human NAD-dependent isocitrate dehydrogenase.

Human NAD-dependent isocitrate dehydrogenase existing as the αβγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory β and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αβ and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of αβγ requires the assembly and cooperative function of both heterodimers. αβγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate αβγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αβ. Furthermore, the allosteric activation of αβγ is through the γ subunit not the β subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme.