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 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 the addition of dithiothreitol. Diamide had no effect on 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.
Garcia
Joshua A
JA
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Minard
Karyl I
KI
Lin
An-Ping
AP
McAlister-Henn
Lee
L
D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006860:Hydrogen Bonding; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D009243:NAD / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004220:Disulfides; D024101:Mitochondrial Proteins; D009243:NAD; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D003545:Cysteine