Disulfide Bond Formation in Yeast NAD+-Specific Isocitrate Dehydrogenase

The tricarboxylic acid cycle NAD(+)-specific isocitrate dehydrogenase (IDH) of Saccharomyces cerevisiae is an octameric enzyme composed of four heterodimers of regulatory IDH I and catalytic IDH2 Subunits. Recent structural analyses revealed the close proximity of Cys-150 residues from IDH2 in adjacent heterodimers, and features of the structure for the ligand-free enzyme suggested that formation of a disulfide bond between these residues might stabilize all inactive form of the enzyme. We constructed two mutant forms of IDH, one containing a C150S Substitution in IDH2 and the other containing C56S/C242S substitutions in IDH2 leaving Cys-150 as the sole cysteine residue. Treatment of the affinity-purified enzymes with diamide resulted in the formation of disulfide bonds and in decreased activities for the wild-type and C56S/C242S enzymes. Both effects were reversible by the addition of dithiothreitol. Diamide had no effect oil the C150S mutant enzyme, suggesting that Cys-150 is essential for the formation of a disulfide bond that inhibits IDH activity. Diamide-Induced formation of the Cys-150 disulfide bond was also observed in vivo for yeast transformants expressing the wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. Finally, natural formation of the Cys-150 disulfide bond with a concomitant decrease in cellular IDH activity was observed during the stationary phase for the parental strain and for transformants expressing wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. A reduction in viability for the latter strain suggests that a decrease in IDH activity is important for metabolic changes in stationary phase cells.