NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is an allosterically regulated enzyme that exists as an octamer composed of two nonidentical subunits, designated IDH1 and IDH2. To determine the contribution of each subunit to regulation and catalysis, a conserved serine residue at the proposed active site of each subunit was mutated to alanine. This mutation in IDH1 resulted in a 6-fold decrease in Vmax and a decrease in cooperativity, but little change in S0.5 for isocitrate. The mutant IDH2, in contrast, exhibited a 60-fold decrease in maximal velocity and a 2-fold reduction in S0.5 for isocitrate, but the cooperativity was unaffected. Responses to the allosteric modifier AMP also differed for the two mutant enzymes. The IDH1 mutant enzyme was not activated by AMP, whereas the IDH2 mutant enzyme exhibited an increase in isocitrate affinity in the presence of AMP similar to that observed with the wild-type enzyme. On the basis of these kinetic 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 individual subunits in yeast strains in which the gene encoding the other subunit had been disrupted. Mitochondrial extracts from strains overexpressing solely IDH1 or IDH2 contained no detectable activity in the 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.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|