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)

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.
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PMID:Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis. 140 Mar 2

The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. 142 Jan 39

The role of the hydroxyl group of tyrosine 6 in the catalytic mechanism of isoenzyme 3-3 of rat glutathione S-transferase has been examined by x-ray crystallography and site-specific replacement of the residue with phenylalanine and evaluation of the catalytic properties of the mutant enzyme. This particuar tyrosine residue is conserved in the sequences of all of the cytosolic enzymes and is found, in crystal structures of both isoenzyme 3-3 from the mu-gene class and an isoenzyme from the pi-gene class, to be proximal to the sulfur of glutathione (GSH) or glutathione sulfonate bound at the active site. The 2.2-A structure of the binary complex of isoenzyme 3-3 and GSH indicates that the hydroxyl group of Tyr6 is located 3.2-3.5 A from the sulfur of GSH, well within hydrogen bonding distance. Removal of the hydroxyl group of Tyr6 has essentially no effect on the dissociation constant (22 +/- 3 microM) for GSH. Nevertheless the Y6F mutant exhibits a turnover number which is only about 1% that of the native enzyme when assayed at pH 6.5 with either 1-chloro-2,4-dinitrobenzene (CDNB) or 4-phenyl-3-buten-2-one. UV difference spectra of the binary enzyme-GSH complexes suggest that the predominant ionization state of GSH in the active site of the Y6F mutant is the neutral thiol (e.g. EY6F.GSH) which is in contrast to the native enzyme in which the thiol is substantially deprotonated (e.g. E.GS-). Spectrophotometric titration suggests that the pKa of the thiol is 6.9 +/- 0.3 in the E.GSH complex and greater than or equal to 8 in the EY6F.GSH binary complex. In addition, the pH dependence of kcat/KmCDNB reveals that the reactions catalyzed by the native enzyme and the Y6F mutant are dependent on a single ionization in the E.GSH and EY6F.GSH complexes with pKa = 6.2 +/- 0.1 and 7.8 +/- 0.3, respectively. The results suggest that the hydrogen bond between Tyr6 and the enzyme-bound nucleophile helps to lower the pKa of GSH in the binary enzyme-substrate complex.
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PMID:Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase. 153 22

Activation of glutathione transferase activity in rat liver microsomes under a variety of conditions producing oxidative stress was investigated. Neither hydrogen peroxide (10 mM) (added or produced endogenously by glucose + glucose oxidase) nor duroquinone together with an NADPH-regenerating system (which generates the superoxide anion radical) had any significant effect on the glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene. On the other hand, incubation of microsomes with 1 mM noradrenaline (which autooxidizes and generates superoxide anion radical) gave a 160% activation, as shown earlier (Aniya and Anders, J Biol Chem 264: 1998-2002, 1989). This was taken as an indication that microsomal glutathione transferase could be activated by oxidative stress. Here, we demonstrate that activation by this compound is due to covalent binding (presumably of the quinone formed during autooxidation). The xanthine/xanthine oxidase system, which generates the superoxide anion radical and hydrogen peroxide, increases microsomal glutathione transferase activity, but this activation was not dependent on the presence of xanthine. Western blots of microsomes treated with xanthine oxidase revealed that activation was due to proteolysis (presumably by contaminating proteases in the xanthine oxidase). In conclusion, there is no firm evidence that rat liver microsomal glutathione transferase is activated directly by reduced oxygen species in the microsomal system. The possibility remains that oxidative stress triggers secondary mechanisms such as generation of reactive intermediates and/or activation of proteolysis, which can in turn increase enzyme activity.
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PMID:Mechanism of activation of rat liver microsomal glutathione transferase by noradrenaline and xanthine oxidase. 157 69

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.
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PMID:Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation. 163 48

We have characterized further the antioxidant responsive element (ARE) identified in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene and the NAD(P)H:quinone reductase gene by mutational and deletion analyses. Our data suggest that the sequence, 5'-puGTGACNNNGC-3' 3'-pyCACTGNNNCG-5' where N is any nucleotide, represents the core sequence of the ARE required for transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds (e.g. beta-naphthoflavone and 3-methylcholanthrene). We also have found that the ARE is responsive to hydrogen peroxide and phenolic antioxidants that undergo redox cycling. These latter data suggest that the ARE is responsive to reactive oxygen species and thus may represent part of a signal transduction pathway that allow eukaryotic cells to sense and respond to oxidative stress.
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PMID:The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. 164 13

As a means to understand the fundamental mechanisms of bleomycin cell killing, we previously isolated 19 bleomycin-sensitive mutants which represent at least six genetically distinct complementation groups (T.D. Stamato, B. Peters, P. Patil, N. Denko, R. Weinstein, and A. Giaccia. Cancer Res., 47: 1588-1592, 1987). One class of mutants represented by the cell line BL-10 displays only hypersensitivity to killing by bleomycin in both acute (16 h) and chronic treatments but no sensitivity to killing by other DNA-damaging agents. Complementation studies between this mutant and human fibroblasts suggested that the human gene which corrects the defect of BL-10 rested on human chromosome 6. It has been reported that the gene for human glutathione S-transferase (GST) alpha also resides on chromosome 6. Measurements of selenium-independent peroxidase (alpha-GST + glutathione peroxidase) activity in wild-type Chinese hamster ovary (CHO) cells, using cumene hydrogen peroxide as a substrate, gave a value of 112 nmol of glutathione oxidized/min/mg protein compared with 88.1 nmol of glutathione oxidized/min/mg protein for BL-10. Measurement of the selenium-dependent peroxidase activity, using H2O2 as a substrate, resulted in 65.9 nmol of reduced glutathione oxidized/min/mg protein in CHO and 81.5 nmol of reduced glutathione oxidized/min/mg protein for BL-10. In other words, BL-10 cells did not exhibit a difference in their ability to metabolize both substrates in contrast to CHO cells. This indicates that BL-10 possesses little alpha-GST activity. Transfection of BL-10 cells with a mammalian expression vector containing the alpha-GST gene increases the survival of BL-10 to bleomycin and does not increase the bleomycin resistance of two other bleomycin mutants which lie in different genetic complementation groups. These data strongly implicate a role for alpha-GST in the resistance of cells to bleomycin.
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PMID:The hypersensitivity of the Chinese hamster ovary variant BL-10 to bleomycin killing is due to a lack of glutathione S-transferase-alpha activity. 171 44

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.
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PMID:Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3. 184 34

Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.
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PMID:Modulation of class Pi glutathione transferase activity by sulfhydryl group modification. 189 44

In seven rabbits subjected to suprarenal aortic coarctation hypertension, the segments above and below the coarctation were tested for the antioxidant defences (i.e. acid-soluble thiol compounds, selenium-dependent and selenium-independent glutathione peroxidase, glutathione reductase, glutathione transferase) and thiobarbituric acid-reactive substances. Seven sham-operated rabbits served as controls. Systolic blood pressure proximal to the ligature increased significantly with respect to pre-operative values after 16 days (117 +/- 8.3 vs 71.7 +/- 5.2 mmHg, P less than 0.05), while pressure distal to the ligature remained normotensive. Higher values of acid-soluble thiol compounds, thiobarbituric acid-reactive substances and increased activities of selenium-dependent glutathione peroxidase, glutathione reductase and glutathione transferase were assayed in the suprarenal with respect to the subrenal segment in both groups. However, the values of the upper segments were more elevated in the experimental group than in controls, but no differences were observed in the lower segments. Glutathione peroxidase activity assayed with cumene hydroperoxide was higher than the activity assayed with hydrogen peroxide in the hypertensive segments, but no differences were detected in the substenotic and control segments. Furthermore, an isoenzymatic form of glutathione transferase, analogous to rat 8-8 glutathione transferase isoenzyme, was detected by immunodiffusion in the hypertensive aorta. The following conclusions may be drawn: (1) a biochemical gradient in glutathione-related enzymes, acid-soluble thiol compounds and thiobarbituric acid-reactive substances between the proximal and distal aorta seems to exist in control rabbits; (2) suprarenal aortic coarctation induces a significant increase in glutathione-related antioxidant defences and thiobarbituric acid-reactive substances of the hypertensive aortic wall.
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PMID:Aortic glutathione-related antioxidant defences in rabbits subjected to suprarenal aortic coarctation hypertension. 194 85


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