The Beta And Gamma Subunits Play Distinct Functional Roles In The Alpha(2)Beta Gamma Heterotetramer Of Human Nad-Dependent Isocitrate Dehydrogenase

Human NAD-dependent isocitrate dehydrogenase existing as the alpha(2)beta gamma heterotetramer, catalyzes the decarboxylation of isocitrate into a-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory beta and gamma subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing alpha beta and alpha gamma heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that alpha beta and alpha gamma alone have considerable basal activity but the full activity of alpha(2)beta gamma requires the assembly and cooperative function of both heterodimers. alpha(2)beta gamma and alpha gamma can be activated by citrate or/and ADP, whereas alpha beta cannot. The binding of citrate or/and ADP decreases the S-0.5,S- isocitrate and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate alpha(2)beta gamma and alpha gamma at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on alpha beta. Furthermore, the allosteric activation of alpha(2)beta gamma is through the. subunit not the gamma subunit. These results demonstrate that the. subunit plays regulatory role to activate the holoenzyme, and the gamma subunit the structural role to facilitate the assembly of the holoenzyme.