KINETIC-ANALYSIS OF NAD-ISOCITRATE DEHYDROGENASE WITH ALTERED ISOCITRATE BINDING-SITES - CONTRIBUTION OF IDH1 AND IDH2 SUBUNITS TO REGULATION AND CATALYSIS()

NAD+-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is an allosterically regulated enzyme that exists as an octamer compos ed of two nonidentical subunits, designated IDH1 and IDH2. To determin e the contribution of each subunit to regulation and catalysis, a cons erved serine residue at the proposed active site of each subunit was m utated to alanine. This mutation in IDH1 resulted in a 6-fold decrease in V(max) and a decrease in cooperativity, but little change in S0.5 for isocitrate. The mutant IDH2, in contrast, exhibited a 60-fold decr ease in maximal velocity and a 2-fold reduction in S0.5 for isocitrate , but the cooperativity was unaffected. Responses to the allosteric mo difier AMP also differed for the two mutant enzymes. The IDH1 mutant e nzyme was not activated by AMP, whereas the IDH2 mutant enzyme exhibit ed an increase in isocitrate affinity in the presence of AMP similar t o that observed with the wild-type enzyme. On the basis of these kinet ic results, a model is presented which proposes that IDH1 functions as a regulatory subunit while IDH2 functions in catalysis. To determine if IDH1 or IDH2 alone is catalytically active, we also expressed the i ndividual subunits in yeast strains in which the gene encoding the oth er subunit had been disrupted. Mitochondrial extracts from strains ove rexpressing solely IDH1 or IDH2 contained no detectable activity in th e presence or absence of AMP. Gel filtration of these extracts showed that both IDH1 and IDH2 behaved as monomers, suggesting that the major subunit interactions within the octamer are between IDH1 and IDH2.