Subunit interactions of yeast NAD(+)-specific isocitrate dehydrogenase

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated I DH1 and IDH2, IDH2 was previously shown to contain the catalytic site, wher eas IDH1 contributes regulatory properties including cooperativity with res pect to isocitrate and allosteric activation by AMP, In this study, interac tions between IDH1 and IDH2 were detected using the yeast two-hybrid system , but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this mo del, based on the three-dimensional structure of the homologous enzyme from Escherichia coil, is that some interactions between subunits occur at isoc itrate binding sites. Based on this model, two residues (Lys-183 and Asp-21 7) in the regulatory IDH1 subunit were predicted to be important in the cat alytic site of IDH2, We found that individually replacing these residues wi th alanine results in mutant enzymes that exhibit a drastic reduction in ca talysis both in vitro and in vivo. Also based on this model, the two analog ous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predi cted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate, A D222A substitution i n IDH2 produces similar regulatory defects and a substantial reduction in V -max in the absence of AMP. Collectively, these results suggest that the ba sic structural/functional unit of yeast isocitrate dehydrogenase is a heter odimer of IDH1 and IDH2 subunits and that each subunit contributes to the i socitrate binding site of the other.