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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inductions of detoxication (phase 2) enzymes, such as glutathione transferases and NAD(P)H:(quinone-acceptor) oxidoreductase, are a major mechanism for protecting animals and their cells against the toxic and neoplastic effects of carcinogens. These inductions result from enhanced transcription, and they are evoked by diverse chemical agents: oxidizable diphenols and phenylenediamines; Michael reaction acceptors; organic isothiocyanates; other electrophiles--e.g., alkyl and aryl halides; metal ions--e.g., HgCl2 and CdCl2; trivalent arsenic derivatives; vicinal dimercaptans; organic hydroperoxides and hydrogen peroxide; and 1,2-dithiole-3-thiones. The molecular mechanisms of these inductions were analyzed with the help of a construct containing a 41-bp enhancer element derived from the 5' upstream region of the mouse liver glutathione transferase Ya subunit gene ligated to the 5' end of the isolated promoter region of this gene, and inserted into a plasmid containing a human growth hormone reporter gene. When this construct was transfected into Hep G2 human hepatoma cells, the concentrations of 28 compounds (from the above classes) required to double growth hormone production, and the concentrations required to double quinone reductase specific activities in Hepa 1c1c7 cells, spanned a range of four orders of magnitude but were closely linearly correlated. Six compounds tested were inactive in both systems. A 26-bp subregion of the above enhancer oligonucleotide (containing the two tandem "AP-1-like" sites but lacking the preceding ETS protein binding sequence) was considerably less responsive to the same inducers. We conclude that the 41-bp enhancer element mediates most, if not all, of the phase 2 enzyme inducer activity of all of these widely different classes of compounds.
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PMID:Chemical and molecular regulation of enzymes that detoxify carcinogens. 838 53

We have previously identified a novel xenobiotic responsive element, which has been termed the antioxidant responsive element (ARE), in the 5'-flanking region of the rat quinone reductase gene (Favreau, L. V., and Pickett, C. B. (1991) J. Biol. Chem. 266, 4556-4561). This element is responsible for basal level expression of the gene as well as transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds. In this communication, we demonstrate that hydrogen peroxide can act as an inducer through the ARE sequence, a phenomenon recently demonstrated for the glutathione S-transferase Ya subunit gene (Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) J. Biol. Chem. 266, 11632-11639). To further characterize the quinone reductase ARE, we demonstrate by DNase I footprinting that in crude Hep G2 nuclear extracts a trans-acting factor exists which interacts with a region of DNA found within the 31-nucleotide ARE sequence. Furthermore, electrophoretic mobility shift assays demonstrate the presence of a specific DNA-protein complex which can be competed only by double-stranded oligonucleotides containing the ARE sequences from the quinone reductase and glutathione S-transferase Ya subunit genes. Methylation interference and protection assays indicate that several guanine residues found in the sequence GTGACTTGGC are involved in the binding of the nuclear factor(s) to the DNA. Although electrophoretic mobility shift assays indicate that the rat quinone reductase ARE does not contain a high affinity recognition site for in vitro translated c-Jun and c-Fos, 12-O-tetradecanoylphorbol 13-acetate can act as an inducer through the ARE sequence in Hep G2 cells.
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PMID:Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-acetate. 839 48

We previously reported that rat glutathione transferase P-form (GST-P) is inactivated by hydrogen peroxide (H2O2). This involves formation of intra- or intersubunit disulfides, at least three extra bands with molecular masses of 21.5, 18, and 37 kDa being exhibited in addition to the native subunit band of 23.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In the present study, GST-P mutants whose cysteine residues were independently substituted with alanine (C14A, C47A, C101A, and C169A) by site-directed mutagenesis were used to identify the cysteine residues responsible for the disulfide bond formation. C14A and C169A were much more inactivated than native GST-P by 1 mM H2O2, whereas C47A and, especially, C101A appeared insensitive to H2O2. On SDS-PAGE, the 21.5-kDa band was not detected in either C47A or C101A. Hydrogen peroxide treatment of mouse GST II, highly homologous to rat GST-P but possessing glycine instead of cysteine at the 101st residue, did not result in generation of the 21.5-kDa band and was also associated with less inactivation. This band was therefore considered to be due to an intrasubunit disulfide bond between Cys-47 and Cys-101. The 37-kDa band was suggested to be due to the formation of intersubunit disulfide bonds between Cys-47 residues in different subunits. Thus the Cys-47 residue together with Cys-101 may be located in an important region for GSH binding, disulfide bond formation between these residues resulting in steric hindrance.
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PMID:Identification of cysteine residues involved in disulfide formation in the inactivation of glutathione transferase P-form by hydrogen peroxide. 842 45

The effect of the administration of three peroxisome-proliferating sulfur-substituted fatty acid analogs on hepatic antioxidant status and lipid peroxidation was studied in rats. After 14 days of treatment, the ratio of induction of peroxisomal fatty acyl-CoA oxidase to catalase was 4.2 and 3.5 in rats treated with 1,10 bis-(carboxymethylthio)decane (BCMTD) and 1-mono (carboxymethylthio)tetradecane (CMTTD), respectively, while the corresponding ratio was 1.3 in 1-mono (carboxyethylthio)tetradecane (CETTD)-treated rats. As compared to the controls an increase in hepatic hydrogen peroxide content was noted in BCMTD- and CMTTD-treated rats, but not CETTD-treated rats. Hepatic lipid peroxidation was increased in all the three treatment groups in a manner not related to the potency of the compounds to induce the peroxisomal hydrogen peroxide metabolizing enzymes. Hepatic glutathione content increased while the activities of its associated enzymes such as glutathione transferase, glutathione peroxidase and glutathione reductase decreased in all the treated rats. Taken together, our data show a relationship between the levels of hydrogen peroxide and lipid peroxidation in rat livers treated with BCMTD and CMTTD. However, increased hepatic lipid peroxidation in CETTD-treated rats cannot be accounted for by the changes in the peroxisomal enzymes.
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PMID:Relationship between peroxisome-proliferating sulfur-substituted fatty acid analogs, hepatic lipid peroxidation and hydrogen peroxide metabolism. 842 18

The evolutionally conserved aspartyl residues (Asp57, Asp98 and Asp152) in human glutathione S-transferase P1-1 were replaced with alanine by site-directed mutagenesis to obtain the mutants (D57A, D98A and D152A). The replacement of Asp98 with alanine resulted in a decrease of the affinity for S-hexyl-GSH-agarose, a 5.5-fold increase of the KmGSH and a 2.9-fold increase of the I50 of S-hexyl-GSH for GSH-CDNB conjugation. Asp98 seems to participate in the binding of GSH through hydrogen bonding with the alpha-carboxylate of the gamma-glutamyl residue of GSH. The kcat of D98A was 2.6-fold smaller than that of the wild-type, and the pKa of the thiol group of GSH bound in D98A was approximately 0.8 pK units higher than those in the wild-type. Asp98 also seems to contribute to the activation of GSH to some extent. On the other hand, most of the kinetic parameters of D57A and D152A were similar to those of the wild-type. However, the thermostabilities of D57A and D152A were significantly lower than that of the wild-type. Asp57 and Asp152 seem to be important for maintaining the proper conformation of the enzyme.
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PMID:Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1. 843 74

To clarify the mechanism of oxidative stress in skeletal muscle atrophied by immobilization, we investigated the change of antioxidant enzyme activities in a typical slow red muscle, the soleus. Atrophied soleus muscles were collected from male Wistar rats (16 weeks old), one ankle joint of which had been immobilized in the fully extended position for 7 days. Also, soleus muscles were collected from intact age-matched rats as control. The activities of Mn-containing superoxide dismutase (Mn-SOD), Cu,Zn-containing superoxide dismutase (Cu,Zn-SOD), Se-dependent glutathione peroxidase (Se-GSHPx), glutathione S-transferase (GST), catalase, and glutathione reductase (GSSGRx) were measured. The activities of Cu,Zn-SOD, GST, and GSSGRx were significantly higher in atrophied muscles, while the others were unchanged. Increased Cu,Zn-SOD and unchanged Mn-SOD levels might reflect increased generation of superoxide anions in the cytoplasm rather than in the mitochondria. Owing to the enhancement of Cu,Zn-SOD and the unaltered Se-GSHPx and catalase activities, hydrogen peroxide is thought to be increased in the cytoplasm. Because there is also an increase of iron in the microsomes of atrophied muscles, the production of hydroxyl radicals, the most aggressive of radicals, might consequently be elevated.
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PMID:Antioxidant enzyme systems in skeletal muscle atrophied by immobilization. 843 91

The crystal structure of the adipocyte lipid-binding protein (ALBP) with coordinated fatty acid shows the hydrophobic ligand bound within a water-filled central cavity with its carboxyl group engaged in a hydrogen bonding network involving, at least in part, the functional groups of residues R126 and Y128. We produced mutant forms of ALBP which altered these amino acids, expressed these in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and examined their ligand-binding properties using the fluorescent fatty acids cis-parinaric acid (c-PA) and 12-(9-anthroyloxy)-oleate (12-AO). The wild-type and all mutated forms of GST-ALBP displayed similar binding affinities for 12-AO, with Kd,app values ranging from 0.5 to 2.4 microM. The binding affinity of ALBP forms R126Q and Y128W for c-PA were reduced about 30-50-fold in comparison to GST-ALBP, while that for the double mutation R126L + Y128F was below the limits of detection. To determine if the hydrogen bonding system functioned in situ, Chinese hamster ovary (CHO) cell transfectants expressing wild-type ALBP demonstrated a moderate (1.5-2-fold) increase in the total rate of [3H]oleate uptake and trafficking into the esterified lipid pools over that of untransfected cells, while the rate of [3H]oleate uptake of the transfected CHOs expressing the R126L + Y128F mutation was identical to that of the control CHOs. In summary, these results suggest that the primary factor contributing to binding affinity of ALBP for fatty acids such as c-PA or oleic acid both in vitro and in situ is the hydrogen bonding network involving at least R126, Y128, and the lipid carboxyl group. However, a ligand with sufficiently large hydrophobic character such as 12-AO can bind in the absence of a functional carboxylate hydrogen bonding network, presumably due to stabilizing entropic interactions with other cavity atoms.
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PMID:Modulation of ligand binding affinity of the adipocyte lipid-binding protein by selective mutation. Analysis in vitro and in situ. 846 12

A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.
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PMID:Ligand effects on the fluorescence properties of tyrosine-9 in alpha 1-1 glutathione S-transferase. 863 25

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2, 4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N. , Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G.(1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.
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PMID:Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase. 866 73

The present study proposes the participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional studies in combination with structural data provide evidence for the alpha-carboxylate of the Glu residue of glutathione acting as a proton acceptor in the catalytic mechanism. The Glu carboxylate is hydrogen-bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu alpha-carboxylate of glutathione is bound in a similar manner to the active sites of mammalian glutathione transferases of classes Alpha, Mu, and Pi, for which three-dimensional structures are known. Mutation of the hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of the Glu residue of glutathione (Thr68->Val) caused a shift of the pH dependence of the enzyme-catalyzed reaction, suggesting that the acidic limb of the pH-activity profile reflects the ionization of the carboxylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two Arg side chains in GST A1-1. One of these residues (Arg45) may influence an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix as a lid over the active site. Removal of the Gly residue from the glutathione molecule caused a 13-fold increase in the KM value for the electrophilic substrate. Thus, the Gly carboxylate of glutathione, by way of influencing the topology of the active site, contributes to the binding of the second substrate of the enzyme. Consequently, the glutathione molecule has several functions in the glutathione transferase catalyzed reactions, not only as a substrate providing the thiol group for different types of chemical reactions but also as a substrate contributing a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second substrate to the enzyme.
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PMID:Involvement of the carboxyl groups of glutathione in the catalytic mechanism of human glutathione transferase A1-1. 867 73


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