Garcia JA, et al. (2009) Disulfide bond formation in yeast NAD+-specific isocitrate dehydrogenase. Biochemistry 48(37):8869-78
Abstract: The tricarboxylic acid cycle NAD<sup>+</sup>-specific isocitrate dehydrogenase (IDH) of <i>Saccharomyces cerevisiae</i> is an octameric enzyme composed of four heterodimers of regulatory IDH1 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 an 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 addition of dithiothreitol. Diamide had no effect on the C150S mutant enzyme, suggesting that Cys-150 is essential for formation of a disulfide bond that inhibits IDH activity. Diamide-induced formation of the Cys-150 disulfide bond was also observed <i>in vivo</i> 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 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.
|Status: Published||Type: Journal Article||PubMed ID: 19645416|
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