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)

Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pKa of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pKa of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on alpha-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of alpha-helix 4 possibly modulates different aspects of the catalytic mechanism of the Lucilia GST.
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PMID:Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione S-transferases. Investigation of Ser-9 and Tyr-113 in a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina. 936 35

The human pi-class glutathione S-transferase (hGST P1-1) is a target for structure-based inhibitor design with the aim of developing drugs that could be used as adjuvants in chemotherapeutic treatment. Here we present seven crystal structures of the enzyme in complex with substrate (glutathione) and two inhibitors (S-hexyl glutathione and gamma-glutamyl- (S-benzyl)cysteinyl-D-phenylglycine). The binding of the modified glutathione inhibitor, gamma-glutamyl-(S-benzyl)cysteinyl-D-phenylglycine, has been characterized with the phenyl group stacking against the benzyl moiety of the inhibitor and making interactions with the active-site residues Phe8 and Trp38. The structure provides an explanation as to why this compound inhibits the pi-class GST much better than the other GST classes. The structure of the enzyme in complex with glutathione has been determined to high resolution (1.9 to 2.2 A) in three different crystal forms and at two different temperatures (100 and 288 K). In one crystal form, the direct hydrogen-bonding interaction between the hydroxyl group of Tyr7, a residue involved in catalysis, and the thiol group of the substrate, glutathione, is broken and replaced by a water molecule that mediates the interaction. The hydrogen-bonding partner of the hydroxyl group of Tyr108, another residue implicated in the catalysis, is space-group dependent. A high-resolution (2.0 A) structure of the enzyme in complex with S-hexyl glutathione in a new crystal form is presented. The enzyme-inhibitor complexes show that the binding of ligand into the electrophilic binding site does not lead to any conformational changes of the protein.
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PMID:The structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution. 939 18

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an "atypical" crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.
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PMID:Solution structure of glutathione bound to human glutathione transferase P1-1: comparison of NMR measurements with the crystal structure. 948 54

We have previously shown that cultured malignant mesothelioma cells contain elevated manganese superoxide dismutase (MnSOD) mRNA levels and activities compared with non-malignant mesothelial cells. As many cytotoxic drugs generate both superoxide and hydrogen peroxide, we assessed the relative significance of catalase and the glutathione redox cycle, as well as glutathione S-transferase (GST), in protecting these cells against hydrogen peroxide and epirubicin toxicity. Mesothelioma cell lines containing high (M38K cells) and low (M14K cells) MnSOD, and non-malignant MeT-5A mesothelial cells were selected for the study. M38K cells were the most resistant of these three cell types to hydrogen peroxide (0.1-0.5 mM, 4 h) and epirubicin (0.1-0.5 microg ml(-1), 48 h) as judged by lactate dehydrogenase (LDH) release and by high-energy nucleotide (ATP, ADP, AMP) depletion. Total glutathione was higher in M38K cells (63.8 +/- 20.3 nnmol mg(-1) protein) than in M14K (25.2 +/- 8.2 nmol mg[-1]) or MeT-5A cells (23.5 +/- 4.5 nmol mg[-1]). Furthermore, GST specific activity was higher in M38K cells (111.3 +/- 15.8 U mg[-1]) than in M14K cells (77.4 +/- 6.6 U mg[-1]) or in MeT-5A cells (68.8 +/- 7.6 U mg[-1]). Western blotting indicated the presence of GST-pi in all these cells, the reactivity again being highest in M38K cells. Depletion of glutathione by buthionine sulphoximine and inhibition of catalase by aminotriazole enhanced hydrogen peroxide toxicity in all cell types, while only the depletion of glutathione increased epirubicin toxicity. We conclude that simultaneous induction of multiple antioxidant enzymes can occur in human mesothelioma cells. In addition to the high MnSOD activity, hydrogen peroxide scavenging antioxidant enzymes, glutathione and GST can partly explain the high hydrogen peroxide and epirubicin resistance of these cells in vitro.
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PMID:Endogenous antioxidant enzymes and glutathione S-transferase in protection of mesothelioma cells against hydrogen peroxide and epirubicin toxicity. 956 45

The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequilin (4-OHEQ)] and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit glutathione S-transferase (GST). Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without the need of oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diadduct. The behavior of 4-OHEQ was found to be more complex than 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene. In contrast, the endogenous catechol estrogens, 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE), were without effect unless tyrosinase was present to convert the catechols to o-quinones. Scavengers of reactive oxygen species and metal chelators had no effect on GST inhibition by catechol estrogens with the exception of the catalase which protected GST activity. Kinetic studies showed that 4-OHEN was a potent irreversible inactivator of GST. Preincubation of the enzyme with 4-OHEN showed a time-dependent increase in inhibitory effect, and gel filtration did not restore GST activity confirming the irreversible nature of the enzyme inactivation. Analysis of the Kitz-Wilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki = 620 microM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 = 7.3 x 10(-)3 s-1). These data suggest that 4-OHEN is an irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. The inhibition mechanism likely involves oxidation of the catechol estrogens to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.
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PMID:Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens. 967 38

Iron nitrilotriacetate (Fe-NTA) is a potent nephrotoxic agent. In this communication we show that Fe-NTA-mediated nephrotoxicity is diminished by 1 wk of oral daily pretreatment of male albino Wistar rats with garlic oil given by gavage at 50 or 100 mg/kg body weight/ml corn oil. Intraperitoneal Fe-NTA treatment at a dose level of 9 mg Fe/kg body weight/10 ml enhances renal microsomal lipid peroxidation and hydrogen peroxide generation which are accompanied by a decrease in the activities of renal antioxidant enzymes (e.g. catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase), and a depletion in the level of renal glutathione. Parallel to these changes, a sharp increase in blood urea nitrogen and serum creatinine has been observed. In addition, Fe-NTA treatment also enhances renal ornithine decarboxylase (ODC) activity and increases [3H]thymidine incorporation into renal DNA. Prophylactic treatment of animals with garlic oil before the administration of Fe-NTA resulted in the diminution of Fe-NTA mediated injury. The enhancement of renal lipid peroxidation and hydrogen peroxide generation was decreased. In addition, there was recovery of glutathione depletion and inhibition of the activities of antioxidant enzymes. Similarly, in animals given the higher dose of garlic oil (100 mg/kg body weight) the enhanced blood urea nitrogen and serum creatinine levels, which are indicative of renal injury, showed a reduction of about 30% and 40%, respectively, in comparison with the group treated with Fe-NTA alone. Pretreatment with garlic oil also ameliorated the Fe-NTA-mediated induction of ODC activity and enhancement of [3H]thymidine incorporation into DNA in a dose-dependent manner. Our data suggest that garlic oil is a potent chemopreventive agent and may suppress Fe-NTA-induced nephrotoxicity.
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PMID:Attenuation of iron-nitrilotriacetate (Fe-NTA)-mediated renal oxidative stress, toxicity and hyperproliferative response by the prophylactic treatment of rats with garlic oil. 967 56

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme-GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.
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PMID:The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism. 967 44

Cytosolic glutathione S-transferase is a family of multi-functional enzymes involved in the detoxification of a large variety of xenobiotic and endobiotic compounds through glutathione conjugation. The three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate, N-(N-L-gamma-glutamyl-3-sulfo-L-alanyl)-glycine, has been determined by the multiple isomorphous replacement method and refined to a crystallographic R factor of 0.183 at 2.1 A resolution. The E. coli enzyme is a globular homodimer with dimensions of 58 Ax56 Ax52 A. Each subunit, consisting of a polypeptide of 201 amino acid residues, is divided into a smaller N-terminal domain (residues 1 to 80) and a larger C-terminal one (residues 89 to 201). The core of the N-terminal domain is constructed by a four-stranded beta-sheet and two alpha-helices, and that of the C-terminal one is constructed by a right-handed bundle of four alpha-helices. Glutathione sulfonate, a competitive inhibitor against glutathione, is bound in a cleft between the N and C-terminal domains. Therefore, the E. coli enzyme conserves overall constructions common to the eukaryotic enzymes, in its polypeptide fold, dimeric assembly, and glutathione-binding site. In the case of the eukaryotic enzymes, tyrosine and serine residues near the N terminus are located in the proximity of the sulfur atom of the bound glutathione, and are proposed to be catalytically essential. In the E. coli enzyme, Tyr5 and Ser11 corresponding to these residues are not involved in the interaction with the inhibitor, although they are located in the vicinity of catalytic site. Instead, Cys10 N and His106 Nepsilon2 atoms are hydrogen-bonded to the sulfonate group of the inhibitor. On the basis of this structural study, Cys10 and His106 are ascribed to the catalytic residues that are distinctive from the family of the eukaryotic enzymes. We propose that glutathione S-transferases have diverged from a common origin and acquired different catalytic apparatuses in the process of evolution.
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PMID:Three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate: catalytic roles of Cys10 and His106. 968 Apr 81

All filariae examined to date express a comprehensive repertoire of both cytoplasmic and secreted anti-oxidant enzymes, although significant differences exist between species and life-cycle stages. Adult Brugia malayi, Dirofilaria immitis and Onchocerca volvulus secrete CuZn superoxide dismutases, and the former two species also secrete a selenocysteine-independent glutathione peroxidase. This enzyme has been localised to the cuticular matrix of B. malayi, and the preferential reduction of fatty acid- and phospholipid hydroperoxides suggests that it may protect cuticular membranes from oxidative damage rather than directly metabolise hydrogen peroxide. Adult O. volvulus may compensate for an apparent deficiency in expression of this enzyme via a secreted variant of glutathione S-transferase. Recent studies have identified a highly expressed family of enzymes collectively termed peroxiredoxins, which most probably play an essential role in reduction of hydroperoxides. Data from cDNA cloning exercises indicate that all filarial species examined thus far express at least two peroxiredoxin variants which have been localised to diverse tissues. In-vitro studies have shown that B. malayi are particularly resistant to oxidative stress, and that the parasites do not rely solely on enzymatic mechanisms of defence. Cuticular lipids are relatively resistant to lipid peroxidation due to the low unsaturation indices of the constituent fatty acyl residues, but complete protection is afforded by the presence of alpha-tocopherol, presumably assimilated from host extracellular fluids. Brugia malayi are also relatively resistant to nitric oxide-mediated toxicity, and this may be due in part to incomplete dependence on aerobic metabolism. Little is known of potential mechanisms for detoxification of nitric oxide derivatives and adaptive responses to oxidative stress in general, and these represent goals for future research.
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PMID:Resistance of filarial nematode parasites to oxidative stress. 977 Jun 16

In order to understand the pulmonary toxicity of ultrafine titanium dioxide (UF-TiO2) particles, various biochemical and chemical parameters were assayed in rat alveolar macrophages (AMs) and cell-free lavage fluid. Single intratracheal exposure of UF-TiO2 (2 mg per rat) caused cytotoxicity to pulmonary AMs. An increase in the population of AMs could be observed, followed by increased activities of lactate dehydrogenase and acid phosphatase in cell-free lavage fluid. In addition, AMs showed an adaptive response because the activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase were increased in these cells. However, this enhancement of antioxidant enzymes could not diminish the enhanced lipid peroxidation and increased rate of hydrogen peroxide generation. The level of glutathione remained decreased in UF-TiO2-exposed rat AMs. The data suggest that the induction of antioxidant enzymes by these cells for self-protection is not sufficient to cope against the toxic action of UF-TiO2, which may lead to oxidative stress.
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PMID:Cytotoxicity, pro-oxidant effects and antioxidant depletion in rat lung alveolar macrophages exposed to ultrafine titanium dioxide. 980 29


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