Purification of yeast isocitrate dehydrogenase.
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ABSTRACT:
SUBMITTER: Illingworth JA
PROVIDER: S-EPMC1174270 | biostudies-other | 1972 Oct
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC2796111 | biostudies-literature
Project description:Mitochondrial NAD(+)-specific isocitrate dehydrogenases (IDHs) are key regulators of flux through biosynthetic and oxidative pathways in response to cellular energy levels. Here we present the first structures of a eukaryotic member of this enzyme family, the allosteric, hetero-octameric, NAD(+)-specific IDH from yeast in three forms: 1) without ligands, 2) with bound analog citrate, and 3) with bound citrate + AMP. The structures reveal the molecular basis for ligand binding to homologous but distinct regulatory and catalytic sites positioned at the interfaces between IDH1 and IDH2 subunits and define pathways of communication between heterodimers and heterotetramers in the hetero-octamer. Disulfide bonds observed at the heterotetrameric interfaces in the unliganded IDH hetero-octamer are reduced in the ligand-bound forms, suggesting a redox regulatory mechanism that may be analogous to the "on-off" regulation of non-allosteric bacterial IDHs via phosphorylation. The results strongly suggest that eukaryotic IDH enzymes are exquisitely tuned to ensure that allosteric activation occurs only when concentrations of isocitrate are elevated.
| S-EPMC2447628 | biostudies-literature
Project description:Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD(+) binding sites, and four AMP binding sites, only half of the sites for each ligand can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2(C150S) octameric enzymes were conducted in the presence of dithiothreitol. These assays demonstrated the presence of eight isocitrate and four AMP binding sites for the wild-type enzyme in the presence of dithiothreitol and for the IDH1/IDH2(C150S) enzyme in the absence or presence of this reagent, suggesting that interactions between sulfhydryl side chains of IDH2 Cys-150 residues limit access to these sites. However, only two NAD(+) sites could be measured for either enzyme. A tetrameric form of IDH (an IDH1(G15D)/IDH2 mutant enzyme) demonstrated half-site binding for isocitrate (two sites) in the absence of dithiothreitol and full-site binding (four sites) in the presence of dithiothreitol. Only one NAD(+) site could be measured for the tetramer under both conditions. In the context of the structure of the enzyme, these results suggest that an observed asymmetry between heterotetramers in the holoenzyme contributes to interactions between IDH2 Cys-150 residues and to half-site binding of isocitrate, but that a form of negative cooperativity may limit access to apparently equivalent NAD(+) binding sites.
| S-EPMC3177981 | biostudies-literature
Project description:Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation with an IDH1 G15D or IDH1 D168K residue substitution produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ∼50% reductions in V(max) and in cooperativity with respect to isocitrate relative to those of the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated (-5)IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit of one tetramer to the IDH2 Cys-150 residues in the other tetramer.
| S-EPMC3137751 | biostudies-literature
Project description:A corrigendum to the article by Hu et al. (2005), Acta Cryst. F61, 486–488. The article by Hu et al. [(2005), Acta Cryst. F61, 486–488] is corrected.
| S-EPMC2242954 | biostudies-literature
Project description:Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four each of regulatory IDH1 and catalytic IDH2 subunits that share 42% sequence identity. IDH2 contains catalytic isocitrate/Mg2+ and NAD+ binding sites whereas IDH1 contains homologous binding sites, respectively, for cooperative binding of isocitrate and for allosteric binding of AMP. Ligand binding is highly ordered in vitro, and IDH exhibits the unusual property of half-site binding for all ligands. The structures of IDH solved in the absence or presence of ligands have shown: (a) a heterodimer to be the basic structural/functional unit of the enzyme, (b) the organization of heterodimers to form tetramer and octamer structures, (c) structural differences that may underlie cooperative and allosteric regulatory mechanisms, and (d) the possibility for formation of a disulfide bond that could reduce catalytic activity. In vivo analyses of mutant enzymes have elucidated the physiological importance of catalytic activity and allosteric regulation of this tricarboxylic acid cycle enzyme. Other studies have established the importance of a disulfide bond in regulation of IDH activity in vivo, as well as contributions of this bond to the property of half-site ligand binding exhibited by the wild-type enzyme.
| S-EPMC3272323 | biostudies-literature
Project description:BackgroundPatients with intrahepatic cholangiocarcinoma (ICC) are prone to recurrence and poor survival. Targeted therapy related to isocitrate dehydrogenase (IDH) is an extremely important treatment. IDH1 and IDH2 mutations are generally thought to have similar effects on the tumor landscape. However, it is doubtful whether these 2 mutations have exactly the same effects on tumor cells and the tumor microenvironment.MethodsAll collected tumor samples were subjected to simultaneous whole-exon sequencing and proteome sequencing.ResultsIDH1 mutations accounted for 12.2%, and IDH2 mutations accounted for 5.5%, all missense mutations. Tumors with IDH mutations had lower proportions of KRAS and TP53 mutations. Mutated genes were obviously enriched in the kinase pathway in the tumors with IDH2 mutations. The signaling pathways were mainly enriched in the activation of cellular metabolic activities and an increase of inhibitory immune cells in the tumors with IDH mutations. Moreover, tumors had unique enrichment in DNA repair in IDH1 mutants and secretion of biological molecules in IDH2 mutants. Inhibitory immune cells might be more prominent in IDH2 mutants, and the expression of immune checkpoints PVR and HLA-DQB1 was more prominent in IDH1 mutants. IDH mutants were more related to metabolism-related and inflammation-immune response clusters, and some belonged to the DNA replication and repair cluster.ConclusionsThese results revealed the differential IDH1 and IDH2 mutation-related landscapes, and we have provided an important reference database to guide ICC treatment.
| S-EPMC11299938 | biostudies-literature
Project description:Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate ?-ketoglutarate (?KG), but recurrent mutations at Arg(132) of IDH1 and Arg(172) of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of ?KG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits ?KG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy.
| S-EPMC4677412 | biostudies-literature
Project description:Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ;isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.
| S-EPMC1174271 | biostudies-other
Project description:Alterations to genes involved in cellular metabolism and epigenetic regulation are implicated in the pathogenesis of myeloid malignancies. Recurring mutations in isocitrate dehydrogenase (IDH) genes are detected in approximately 20% of adult patients with acute myeloid leukemia (AML) and 5% of adults with myelodysplastic syndromes (MDS). IDH proteins are homodimeric enzymes involved in diverse cellular processes, including adaptation to hypoxia, histone demethylation and DNA modification. The IDH2 protein is localized in the mitochondria and is a critical component of the tricarboxylic acid (also called the 'citric acid' or Krebs) cycle. Both IDH2 and IDH1 (localized in the cytoplasm) proteins catalyze the oxidative decarboxylation of isocitrate to ?-ketoglutarate (?-KG). Mutant IDH enzymes have neomorphic activity and catalyze reduction of ?-KG to the (R) enantiomer of 2-hydroxyglutarate, which is associated with DNA and histone hypermethylation, altered gene expression and blocked differentiation of hematopoietic progenitor cells. The prognostic significance of mutant IDH (mIDH) is controversial but appears to be influenced by co-mutational status and the specific location of the mutation (IDH1-R132, IDH2-R140, IDH2-R172). Treatments specifically or indirectly targeted to mIDH are currently under clinical investigation; these therapies have been generally well tolerated and, when used as single agents, have shown promise for inducing responses in some mIDH patients when used as first-line treatment or in relapsed or refractory AML or MDS. Use of mIDH inhibitors in combination with drugs with non-overlapping mechanisms of action is especially promising, as such regimens may address the clonal heterogeneity and the multifactorial pathogenic processes involved in mIDH myeloid malignancies. Advances in mutational analysis have made testing more rapid and convenient, and less expensive; such testing should become part of routine diagnostic workup and repeated at relapse to identify patients who may benefit from treatments that target mIDH.
| S-EPMC5292675 | biostudies-literature