Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.41 (isocitrate dehydrogenase)
3,101 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.
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PMID:Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation. 1 68

Electron paramagnetic resonance (EPR) spectra were obtained for various isocitrate dehydrogenase-Mn(II) complexes. The qualitative effects of the binding of substrates, nucleotides, and substrate analogues on the isotropic character of the electronic environment of enzyme-bound Mn(II) were subsequently investigated. The addition of isocitrate produces a markedly anisotropic spectrum whereas alpha-ketoglutarate does not alter the spectrum of enzyme-Mn(II) substantially. This suggests direct coordination of isocitrate to the Mn(II) but perphaps a different mode of binding for alpha-ketoglutarate. Other studies demonstrated mutually exclusive binding relationships between TPN and TPNH, between Mn-isocitrate and TPNH, and between HCO3-(CO2) and formate or thiocyanate. Indirect evidence supporting CO2 rather than HCO3-as the actual reactive species which binds to the enzyme in the reductive carboxylation reaction is presented on the basis of the results of the formate and thiocyanate studies. From the EPR results recorded for ternary, quaternary, and quinary enzyme-substrate complexes, correlations between the appearance of fine structure signals and the binding of individual substrates and/or nucleotides are found, and tentative assignments of such signals are made on this basis. Additional studies were conducted to determine binding constants for Mg(II) Co(II), and Co-isocitrate, and a comparison was made with kinetically determined binding constants.
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PMID:Structure-function relationships in TPN-dependent isocitrate dehydrogenase. I. Electron paramagnetic resonance studies of the interaction of enzyme-bound Mn(II) with substrates, cofactors, and substrate analogues. 1 44

D-Garcinia acid (D-threo-1,2-dihydroxy-1,2,3-propanetricarboxylate), like D-isocitrate, has an alpha-DS-hydroxyl group and a beta-LS configuration of the second carboxyl group. The maximal velocity of pyridine nucleotide reduction with D-garcinia acid is 8 and 21% of D-threo-isocitrate with the DPN-linked and TPN-linked isocitrate dehydrogenase from bovine heart, respectively. The other stereoisomers of hydroxycitrate [L-garcinia acid, D- and L-hibiscus acid (D- and L-erythro-1,2-dihydroxy-1,2,3-propanetricarboxylate)] are inactive. DL-threo-Homoisocitrate (DL-threo-1-hydroxy-1,2,4-butanetricarboxylate) supports DPN+ reduction at 10-15% of the rate observed for isocitrate with the DPN-specific enzyme, but is not a substrate for TPN-linked isocitrate dehydrogenase. The values of apparent S0.5 for total isocitrate and total garcinia acid are similar with both enzymes; the apparent S0.5 of total homoisocitrate is two- to threefold higher than that of total isocitrate with the DPN-linked enzyme. Enzymatic oxidative decarboxylation of garcinia acid and homoisocitrate leads to formation of alpha-keto-beta-hydroxyglutarate and alpha-ketoadipate, respectively. DL-Methylmalate (DL-1-hydroxy-2-methylsuccinate) is inactive as a substrate for either dehydrogenase as are the newly synthesized compounds: DL-threo-gamma-isocitrate amide (DL-threo-1-hydroxy-3-carbamy01,2-propanedicarboxylate), beta-methyl-DL-isocitrate (DL-1-hydroxy-2-methyl-1,2,3-propanetricarboxylate), beta-methyl-DL-garcinia acid (DL-threo-1-hydroxyl-2-methoxy-1,2,3-propanetricarboxylate), DL-1-hydroxyl-1,2,2-ethanetricarboxylate, and DL-1,4-dihydroxy-1,2-butanedicarboxylate.
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PMID:Substrate activity of structural analogs of isocitrate for isocitrate dehydrogenases from bovine heart. 23 63

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.
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PMID:Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver. 23 45

The binding of TPNH to native and chemically modified pig heart TPN-dependent isocitrate dehydrogenase was studied by the techniques of ultrafiltration and fluorescence enhancement. A single site (per peptide chain) was found for TPNH with a dissociation constant (KD = 1.45 muM) that is quantitatively comparable to the Michaelis constant. The oxidized coenzyme, TPN+, weakens the binding of TPNH. The substrate manganous isocitrate also inhibits the binding of TPNH and, reciprocally, TPNH inhibits the binding of manganous isocitrate, suggesting that binding to the reduced coenzyme and substrate sites is mutually exclusive. Ultrafiltration experiments with carbonyl [14C]TPN+ revealed the existence of two sites with a dissociation constant (49 muM) more than ten times higher than the Michaelis constant. This observation excludes a random mechanism for isocitrate dehydrogenase or a sequential mechanism in which TPN+ binds first. Four chemically modified isocitrate dehydrogenases have been prepared: enzyme inactivated by reaction of a single methionyl residue with iodoacetate, by modification of a glutamyl residue by glycinamide (in the presence of a water soluble carbodiimide), by reaction of four cysteines successively with 5,5'-dithiobis(2-nitrobenzoic acid) and potassium cyanide, or by addition of two cysteine residues to N-ethylmaleimide. These enzymes were tested for their ability to bind TPN+, TPNH, and manganous isocitrate. In the cases of the cysteinyl and glutamyl-modified enzymes, inactivation appears to be due primarily to loss of the ability to bind the substrate manganous isocitrate. In constrast, the methionyl residue may participate in the coenzyme binding site or, more likely, may be involved in a step in catalysis subsequent to binding.
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PMID:Coenzyme binding by native and chemically modified pig heart triphosphopyridine nucleotide dependent isocitrate dehydrogenase. 24 96

The specific activity of a peroxisomal enzyme, lactate oxidase, and of pyruvate kinase and lactate dehydrogenase, which are not peroxisomal, increased rapidly when shaken cultures of Tetrahymena were transferred to conditions of oxygen restriction and supplemented with glucose. Two other peroxisomal enzymes, catalase and TPN-linked isocitrate dehydrogenase, did not increase substantially, nor did succinate dehydrogenase. The increases were reduced if glucose was not added at the time of transfer, and were prevented by actinomycin D or cycloheximide, but not by chloramphenicol. The results suggest an involvement of peroxisomes in the metabolism of glycolytic endproducts when the availability of oxygen to the cell is limiting.
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PMID:Synthesis of glycolytic and peroxisomal enzymes in Tetrahymena following a change in culture conditions. 80 70

This paper presents preliminary data concerning the relationship of various components of glandular epithelium and effect of enzymes on metabolism, storage, and release of certain substances in normal and abnormal endometria. Activity of these endometrial enzymes has been compared between two groups: 252 patients with normal menstrual histories and 156 patients, all over the age of 40, with abnormal uterine bleeding. Material was obtained by endometrial biopsy or curettage. In the pathologic classification of the group of 156, 30 patients had secretory endometria, 88 patients had endometria classified as proliferative, 24 were classified as endometrial hyperplasia, and 14 were classified as adenocarcinoma. All tissue was studied by histologic, histochemical, and biochemical methods. Glycogen synthetase activity caused synthesis of glucose to glycogen, increasing in amount until midcycle, when glycogen phosphorylase activity caused the breakdown to glucose during the regressive stage of endometrial activity. This normal cyclic activity did not occur in the abnormal endometria, where activity of both enzymes continued at low constant tempo. Only the I form of glycogen synthetase increased as the tissue became more hyperplastic. With the constant glycogen content and the increased activity of both the TPN isocitric dehydrogenase and glucose-6-phosphate dehydrogenase in the hyperplastic and cancerous endometria, tissue energy was created, resulting in abnormal cell proliferation. These altered biochemical and cellular activities may be the basis for malignant cell growth.
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PMID:The effect of enzymes upon metabolism, storage, and release of carbohydrates in normal and abnormal endometria. 81 24

DL-threo-alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a substrate for bovine heart aconitase and an inhibitor of TPN-linked isocitrate dehydrogenase from liver and heart. The isomer of alpha-methylisocitrate formed from alpha-methyl-cis-aconitate (cis-2-butane-1,2,3-tricarboxylate) by aconitase inhibits TPN-linked isocitrate dehydrogenase and has been identified as D-threo-alpha-methylisocitrate (2S,3R)-3-hydroxy-1,2,3-butanetricarboxylate) by optical rotation and circular dichroism studies. Mitochondrial bovine heart aconitase catalyzes a reversible reaction between D-threo-alpha-methylisocitrate (Km, 0.2 mM) and alpha-methyl-cis-aconitate (Km, 0.05 mM) at pH 7.4. However, formation of methylcitrate (2-hydroxy-1,2,3-butanetricarboxylate) from these substrates or utilization of synthetic methylcitrate for formation of these products could not be demonstrated with bovine heart aconitase. DL-threo-alpha-Methylisocitrate is also a substrate for aconitase from rat liver cytosol (Km, 0.1 mM); Vmax with citrate is approximately 1.4 times that with DL-threo-alpha-methylisocitrate. The ratio of activities for these substrates observed with the bovine heart enzyme is about 5. Formation of alpha-methyl-cis-aconitate from synthetic methylcitrate could not be detected spectrophotometrically with the liver aconitase; if it occurs with either the liver or the heart enzyme, the rate would be less than 0.1% that obtained with DL-threo-alpha-methylisocitrate. A new synthesis of methylcitric acid in good yields from diethyl alpha-methyl-beta-ketoglutarate (diethyl 2-methyl-3-oxoglutarate) and cyanide has been described. NMR spectroscopy indicates that this synthetic methylcitric acid contains the two racemic pairs of diastereoisomers.
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PMID:Identification of D-threo-alpha-methylisocitrate as stereochemically specific substrate for bovine heart aconitase and inhibitor of TPN-linked isocitrate dehydrogenase. 85 1

Spectroscopic, ultrafiltration, and kinetic studies have been used to characterize interactions of reduced and oxidized triphosphopyridine nucleotides (TPNH and TPN), 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P), and adenosine 2',5'-bisphosphate [Ado(2',5')P2] with with TPN-specific isocitrate dehydrogenase. Close similarity of the UV difference spectra and of the protein fluorescence changes accompanying the formation of the binary complexes provides evidence for the binding of these nucleotides to the same site on the enzyme. From the pH dependence of the dissociation constants for TPNH binding to TPN-specific isocitrate dehydrogenase in the absence and in the presence of Mn2+, over the pH range 5.8-7.6, it has been demonstrated that the nucleotide binds to the enzyme in its unprotonated, metal-free form. The involvement of positively charged residues, protonated over the pH range studied, has been postulated. One TPNH binding site per enzyme subunit has been measured by fluorescence and difference absorption titrations. A dramatic effect of ionic strength on binding has been demonstrated: about a 1000-fold decrease in the dissociation constant for TPNH has been observed at pH 7.6 upon decreasing ionic strength from 0.336 (Kd = 1.2 +/- 0.2 microM) to 0.036 M (Kd = 0.4 +/- 0.1 nM) in the presence and in the absence of 100 mM Na2SO4, respectively. Weak competition of sulfate ions for the nucleotide binding site has been observed (KI = 57 +/- 3 mM). The binding of TPN in the presence of 100 mM Na2SO4 at pH 7.6 is about 100-fold weaker (Kd = 110 +/- 22 microM) than the binding of the reduced coenzyme and is similarly affected by ionic strength. These results demonstrate the importance of electrostatic interactions in the binding of the coenzyme to TPN-specific isocitrate dehydrogenase. The large enhancement of protein fluorescence caused by binding of TPN and Rib-P2-Ado-P (delta Fmax = 50%) and of Ado(2',5')P2 (delta Fmax = 41%) has been ascribed to a local conformational change of the enzyme. An apparent stoichiometry of 0.5 nucleotide binding site per peptide chain was determined for TPN, Rib-P2-Ado-P, and Ado(2',5')P2 from fluorescence titrations, in contrast to one binding site per enzyme subunit determined from UV difference spectral titration and ultrafiltration experiments. Thus, the binding of one molecule of the nucleotide per dimeric enzyme molecule is responsible for the total increase in protein fluorescence, while binding to the second subunit does not cause further change.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Spectroscopic studies of the interactions of coenzymes and coenzyme fragments with pig heart, oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase. 384 32

Catalytic reaction of the 2', 3'-dialdehyde analog of TPN (oTPN) with pig heart TPN-dependent isocitrate dehydrogenase in the presence of the substrate manganous isocitrate results in the formation of the dialdehyde derivative of TPNH (oTPNH). In the absence of the substrate, modification by oTPN leads to a progressive inactivation of the enzyme. The dependence of the pseudo-first order rate constants on the reagent concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification (kmax = 5.5 X 10(-2) min-1; K1 = 290 microM). Reaction of [14C]oTPN with the enzyme results in the incorporation of 2 mol of oTPN/mol of peptide chain. No appreciable protection against either inactivation or incorporation by the natural ligands TPN and TPNH was obtained, suggesting different modes of binding of the analog in the presence and absence of the substrate isocitrate. Enzymatically synthesized oTPNH has been isolated and demonstrated to act as an affinity label for a TPNH-binding site of isocitrate dehydrogenase. The inactivation process exhibits saturation kinetics (kmax = 2.67 X 10(-3) min-1; K1 = 33 microM). Protection against activity loss, as well as a decrease in incorporation from 2 to 1 eq of [14C]oTPNH bound/peptide chain was observed in the presence of 1 mM TPNH. From the TPNH concentration dependence of the inactivation rate by oTPNH, a dissociation constant of 3.4 microM is calculated for TPNH, indicating binding of the analog to a specific TPNH-binding site on the enzyme. Although dialdehyde derivatives are frequently assumed to form Schiff bases with proteins, the evidence presented suggests the formation of morpholino derivatives as the products of the covalent reaction of isocitrate dehydrogenase with the dialdehyde derivatives of TPN and TPNH. The new reagent, oTPNH, may serve as an affinity label for other dehydrogenases.
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PMID:Modification of TPN-dependent isocitrate dehydrogenase by the 2', 3'-dialdehyde derivatives of TPNH and TPN. 687 92


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