Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

Gabriel, Jerome L.; Zervos, Paula R.; Plaut, G.W.E.

doi:10.1016/0026-0495(86)90175-7

Cited by 75 publications

(66 citation statements)

References 31 publications

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“…Similar functional differentiation through hetero-complex formation between paralogues is found in isocitrate dehydrogenase (IDH) from Saccharomyces cerevisiae (Barnes et al, 1971). In S. cerevisiae, IDH1 functions as the catalytic subunit and IDH2 functions as the regulatory subunit by forming an a 4 b 4 -type hetero-octameric structure (Gabriel et al, 1986). In that system, hetero-complex formation has the advantage of allowing growth in acetate medium (Hu et al, 2006).…”

Section: Discussionmentioning

confidence: 82%

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Tanaka

Miyazaki

et al. 2010

Microbiology

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An extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes, gdhA and gdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA-GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA-GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1 mM leucine elevated the GDH activity of the co-purified GdhA-GdhB by 974 and 245 % for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a heterooligomeric GDH that can be regulated allosterically.

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Section: Discussionmentioning

confidence: 82%

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Tanaka

Miyazaki

et al. 2010

Microbiology

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“…All metabolites were analyzed as tert-butyldimethylsilyl derivatives on a Hewlett-Packard 5890 series II plus GC coupled to a 5972 mass-selective detector equipped with an HP-5 fused silica capillary column (50 m, 0.2-mm inner diameter, 0.33-m film thickness). Concentrations of CAC intermediates were determined in tissue samples spiked with [1,[5][6][7][8][9][10][11][12][13] Other assays. PO 2, PCO2, free Ca 2ϩ , and pH were determined in influent and effluent perfusates collected under normoxia (18 min) or LFI (88 min) using a pH, blood gas, and electrolyte analyzer (ABL 70 series, Radiometer; Copenhagen).…”

Section: Gc-msmentioning

confidence: 99%

“…This is in contrast with NAD ϩ -ICDH, which has kinetic properties that are consistent with its unidirectional operation in vivo toward ␣-KG formation. The latter enzyme is highly regulated by a variety of positive (Ca 2ϩ , ADP, and citrate) and negative (ATP, NADH, and NADPH) effectors (10).…”

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confidence: 99%

Reverse flux through cardiac NADP⁺-isocitrate dehydrogenase under normoxia and ischemia

Comte¹,

Vincent

Bouchard³

et al. 2002

American Journal of Physiology-Heart and Circulatory Physiology

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Comte, Blandine, Geneviè ve Vincent, Bertrand Bouchard, Mohamed Benderdour, and Christine Des Rosiers. Reverse flux through cardiac NADP ϩ -isocitrate dehydrogenase under normoxia and ischemia. Am J Physiol Heart Circ Physiol 283: H1505-H1514, 2002. First published June 6, 2002 10.1152/ajpheart.00287.2002Little is known about the role of mitochondrial NADP ϩ -isocitrate dehydrogenase (NADP ϩ -ICDH) in the heart, where this enzyme shows its highest expression and activity. We tested the hypothesis that in the heart, NADP ϩ -ICDH operates in the reverse direction of the citric acid cycle (CAC) and thereby may contribute to the fine regulation of CAC activity (Sazanov and Jackson, FEBS Lett 344: 109-116, 1994). We documented a reverse flux through this enzyme in rat hearts perfused with the medium-chain fatty acid octanoate using [U-13 C5]glutamate and mass isotopomer analysis of tissue citrate (Comte et al., J Biol Chem 272: 26117-26124, 1997). In this study, we assessed the significance of our previous finding by perfusing hearts with long-chain fatty acids and tested the effects of changes in O2 supply. We showed that under all of these conditions citrate was enriched in an isotopomer containing five 13 C atoms. This isotopomer can only be explained by substrate flux through reversal of the NADP ϩ -ICDH reaction, which is evaluated at 3-7% of flux through citrate synthase. Small variations in reversal fluxes induced by low-flow ischemia that mimicked hibernation occurred despite major changes in contractile function and O2 consumption of the heart as well as citrate and succinate release rates and tissue levels. Our data show a reverse flux through NADP ϩ -ICDH and support its hypothesized role in the fine regulation of CAC activity in the normoxic and O2-deprived heart. citric acid cycle; citrate release; isotopomer analysis; 13 C substrate; anaplerosis CARDIAC ISCHEMIC DISEASES have been associated with chronic alterations of energy metabolism such as increased myocardial citrate release (24, 42). The cause of this deregulated cardiac citrate metabolism is unclear. Myocardial citrate release reflects its efflux from mitochondria (43) where it is synthesized by citrate synthase during normal operation of the citric acid cycle (CAC; Fig. 1). Citrate release, which is modulated by substrates and/or O 2 supply (24, 28, 29, 43), represents at most 1% of CAC flux. Mitochondrial citrate efflux appears normally to be compensated largely by flux through anaplerotic reactions such as pyruvate carboxylation, which represents between 2 and 8% of CAC flux (5,28,29).Citrate synthesis could also occur through a reductive process, which involves the participation of the CAC enzymes aconitase and NADP ϩ -linked isocitrate dehydrogenase (NADP ϩ -ICDH). Aconitase catalyzes the reversible interconversion between citrate and isocitrate. Its activity is modulated by oxidative stress (3,25). NADP ϩ -ICDH catalyzes the reversible interconversion between isocitrate and ␣-ketoglutarate (␣-KG). It has no known allosteric effector. This...

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“…The affinities of the yeast and human enzymes (IDH, 5 EC 1.1.1.41) for isocitrate are allosterically regulated positively by AMP and ADP, respectively, and the human enzyme is also regulated negatively by ATP (1,2). Both enzymes are negatively regulated by NADH (3,4). This control has been proposed to contribute to inverse regulation of rates of energy production by oxidative pathways and by glycolysis (5 Yeast IDH is a hetero-octamer composed of four IDH1 (M r ϭ 38,001) and four IDH2 (M r ϭ 37,755) subunits with 42% sequence identity (6 -8) (supplemental Fig.…”

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confidence: 99%

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Taylor

Hart

et al. 2008

Journal of Biological Chemistry

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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.The oxidative decarboxylation of isocitrate to ␣-ketoglutarate catalyzed by mitochondrial NAD ϩ -specific isocitrate dehydrogenases is a rate-limiting step in the tricarboxylic acid cycle. The affinities of the yeast and human enzymes (IDH, 5 EC 1.1.1.41) for isocitrate are allosterically regulated positively by AMP and ADP, respectively, and the human enzyme is also regulated negatively by ATP (1, 2). Both enzymes are negatively regulated by NADH (3, 4). This control has been proposed to contribute to inverse regulation of rates of energy production by oxidative pathways and by glycolysis (5) Yeast IDH is a hetero-octamer composed of four IDH1 (M r ϭ 38,001) and four IDH2 (M r ϭ 37,755) subunits with 42% sequence identity (6 -8) (supplemental Fig. 1). Results of targeted mutagenesis studies (9 -13) suggest that IDH2 contains a catalytic isocitrate/Mg 2ϩ -and NAD ϩ -binding site, whereas the homologous site in IDH1 functions in cooperative binding of isocitrate and in binding of the allosteric activator AMP. Yeast two-hybrid assays and other mutagenesis studies (13, 14) strongly suggest that a heterodimer of IDH1 and IDH2 is the fundamental structural building block of the enzyme and that IDH1 contributes a few residues to catalytic sites in IDH2, while the analogous residues of IDH2 function in the regulatory ligand-binding sites in IDH1. Visualization of subunit interactions within and between heterodimers in the holoenzyme is essential to define the molecular basis for allosteric communication between such sites and to provide information prerequisite for an understanding of the evolution of the highly regulated allosteric IDH hetero-octamer from the more ancient nonallosteric, homodimeric bacterial IDH enzymes. Mammalian NAD ϩ -specific isocitrate dehydrogenases are structurally more complex octamers than yeast IDH, containing three subunits in a ratio of ␣ 2 :␤:␥, with the ␣-s...

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Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

Cited by 75 publications

References 31 publications

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Reverse flux through cardiac NADP⁺-isocitrate dehydrogenase under normoxia and ischemia

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

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Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

Cited by 75 publications

References 31 publications

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Reverse flux through cardiac NADP+-isocitrate dehydrogenase under normoxia and ischemia

Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

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Reverse flux through cardiac NADP⁺-isocitrate dehydrogenase under normoxia and ischemia