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 glutathione S-transferase (GST) isozyme A1-1 contains at its active site a catalytic tyrosine, Tyr9, which hydrogen bonds to, and stabilizes, the thiolate form of glutathione, GS-. In the substrate-free GST A1-1, the Tyr 9 has an unusually low pKa, approximately 8.2, for which the ionization to tyrosinate is monitored conveniently by UV and fluorescence spectroscopy in the tryptophan-free mutant, W21F. In addition, a short alpha-helix, residues 208-222, provides part of the GSH and hydrophobic ligand binding sites, and the helix becomes "disordered" in the absence of ligands. Here, hydrostatic pressure has been used to probe the conformational dynamics of the C-terminal helix, which are apparently linked to Tyr 9 ionization. The extent of ionization of Tyr 9 at pH 7.6 is increased dramatically at low pressures (p1/2 = 0.52 kbar), based on fluorescence titration of Tyr 9. The mutant protein W21F:Y9F exhibits no changes in tyrosine fluorescence up to 1.2 kbar; pressure specifically ionizes Tyr 9. The volume change, delta V, for the pressure-dependent ionization of Tyr 9 at pH 7.6, 19 degrees C, was -33 +/- 3 mL/mol. In contrast, N-acetyl tyrosine exhibits a delta V for deprotonation of -11 +/- 1 mL/mol, beginning from the same extent of initial ionization, pH 9.5. The pressure-dependent ionization is completely reversible for both Tyr 9 and N-acetyl tyrosine. Addition of S-methyl GSH converted the "soft" active site to a noncompressible site that exhibited negligible pressure-dependent ionization of Tyr 9 below 0.8 kbar. In addition, Phe 220 forms part of an "aromatic cluster" with Tyr 9 and Phe 10, and interactions among these residues were hypothesized to control the order of the C-terminal helix. The amino acid substitutions F220Y, F2201, and F220L afford proteins that undergo pressure-dependent ionization of Tyr 9 with delta V values of 31 +/- 2 mL/mol, 43 +/- 3 mL/mol, and 29 +/- 2 mL/mol, respectively. The p1/2 values for Tyr 9 ionization were 0.61 kbar, 0.41 kbar, and 0.46 kbar for F220Y, F220I, and F220L, respectively. Together, the results suggest that the C-terminal helix is conformationally heterogeneous in the absence of ligands. The conformations differ little in free energy, but they are significantly different in volume, and mutations at Phe 220 control the conformational distribution.
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PMID:Pressure-dependent ionization of Tyr 9 in glutathione S-transferase A1-1: contribution of the C-terminal helix to a "soft" active site. 909 97

We have expressed, purified, and analyzed the iron-containing superoxide dismutase (FeSOD) of Escherichia coli with mutations directed at tyrosine position 34 to introduce phenylalanine (SODY34F), serine (SODY34S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified from SOD-deficient cells using a GST-FeSOD fusion protein intermediate which was subsequently cleaved with thrombin and repurified. Specific activities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually inactivates the enzyme (42 u/mg); mutation to cysteine greatly reduces activity (563 u/mg), but the SODY34F mutant retains nearly 40% of the activity of wild type (1205 u/mg). Fusion protein intermediates were also shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expressed mutant enzymes. SODY34F exhibited decreased thermal stability, reduced activity at high pH, and a pronounced increase in sensitivity to the inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a structural role (probably through hydrogen bonding to glutamine at position 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the active site of both substrate and inhibitor molecules.
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PMID:The conserved residue tyrosine 34 is essential for maximal activity of iron-superoxide dismutase from Escherichia coli. 912 14

The possible role of the hydroxyl group of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1 has been investigated by means of site-directed mutagenesis, steady-state kinetic analysis, and crystallographic studies. Three representative cosubstrates have been used, i.e. ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and 1-chloro-2,4-dinitrobenzene. In the presence of ethacrynic acid, the enzyme follows a rapid equilibrium random bi-bi mechanism with a rate-limiting step which occurs after the addition of the substrates and before the release of products. The replacement of Tyr 108 with Phe yields a 14-fold decrease of k(cat), while it does not change appreciably the affinity of the H site for the substrate. In this case, it would appear that the role of the hydroxyl function is to stabilize the transition state for the chemical step, i.e. the Michael addition of GSH to the electrophilic substrate. Crystallographic data are compatible with this conclusion showing the hydroxyl group of Y108 in hydrogen bonding distance of the ketone moiety of ethacrynic acid [Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., & Parker, M. W. (1997) Biochemistry 36, 576-585]. Moreover, no structural differences are observed between the Y108F mutant and the wild type, suggesting that the removal of the hydroxyl group is solely responsible for the loss of activity. A different involvement of Tyr 108 appears in the catalyzed conjugation of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole with GSH in which the rate-limiting step is of a physical nature, probably a structural transition of the ternary complex. The substitution of Tyr 108 yields an approximately 7-fold increase of k(cat) and a constant k(cat)/Km(NBD-Cl) value. Lack of a critical hydrogen bond between 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and Tyr 108 appears to be the basis of the increased k(cat). In the 1-chloro-2,4-dinitrobenzene/GSH system, no appreciable changes of kinetics parameters are found in the Y108F mutant. We conclude that Y108 has a multifunctional role in glutathione transferase P1-1 catalysis, depending on the nature of the electrophilic cosubstrate.
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PMID:Multifunctional role of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1. Crystallographic and kinetic studies on the Y108F mutant enzyme. 916 93

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is the second identified Se-dependent intracellular glutathione peroxidase (PHGPX) that reduces phospholipid hydroperoxides. The objective of this study was to determine the developmental regulation of PHGPX expression in tissues of neonatal, weanling and finishing pigs (Sus scrofa) compared with the expression of the classic Se-dependent cellular glutathione peroxidase (GPX) and the Se-independent enzyme, glutathione S-transferase (GST). Eight different tissues were collected from Se-adequate male pigs aged 1, 28 and 180 days, and supernatant of the tissue homogenate was assayed for PHGPX, GPX and GST activities by using phosphatidylcholine hydroperoxide, hydrogen peroxide and 1-chloro-2,4-dinitrobenzene as substrate, respectively. Total RNA was isolated from four tissues and assayed for PHGPX mRNA expression. Both mRNA and activity expression of PHGPX in most assayed tissues was increased as pigs became older (P < 0.05), but increases in PHGPX mRNA levels between ages did not fully account for all changes in activity. Expression of GPX activity was increased more than that of PHGPX between day 1 and day 28 (P < 0.0001). Expression of GST activity in various tissues was also affected by age (P < 0.01) but lacked a consistent relationship with the changes in GPX and PHGPX activity. Tissue-specific patterns of developmental expression of these enzymes may be related to the susceptibility of organs to pro-oxidant injuries. In conclusion, expression of PHGPX mRNA and activity in various tissues of pigs is developmentally increased over ages, and the pattern is somewhat different from that of GPX.
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PMID:Comparison of age-related differences in expression of phospholipid hydroperoxide glutathione peroxidase mRNA and activity in various tissues of pigs. 918 19

A three-dimensional structural model of the dichloromethane dehalogenase (DCMD) from Methylophilus sp. DM11 is constructed based on sequence similarities to the glutathione S-transferases (GSTs). To maximize sequence identity and minimize gaps in the alignment, a hybrid approach is used that takes advantage of the increased homology found between DM11 and domain I of the sheep blowfly theta class GST (residues 1-79) and domain II of the human alpha class GST (residues 81-222). The resulting structure has C alpha root mean square deviations of 1.16 A in domain I and 1.83 A in domain II from the template GSTs, which compare well to those seen in other GST inter-class comparisons. The model is further applied to explore the structural basis for substrate binding and catalysis. A conserved network of hydrogen bonds is described that binds glutathione to the G site, placing the thiol group in a suitable location for nucleophilic attack of dichloromethane. A mechanism is proposed that involves activation through a hydrogen bond interaction between Ser12 and glutathione, similar to that found in the theta-GSTs. The model also demonstrates how aromatic residues in the hydrophobic site (H site) could play a role in promoting catalysis: His116 and Trp117 are ideally situated to accept a growing negative charge on a chlorine of dichloromethane, stabilizing displacement. This scheme is consistent with experimental results of single-point mutations and comparisons with other GST structures and mechanisms.
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PMID:Knowledge-based modeling of a bacterial dichloromethane dehalogenase. 918 39

Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.
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PMID:Structure and function of the xenobiotic substrate-binding site and location of a potential non-substrate-binding site in a class pi glutathione S-transferase. 924 1

Theoretical calculations were performed to examine the ionization of the phenolic group of Tyr7 and the thiol group of glutathione in aqueous solution and in the protein class-pi glutathione S-transferase (GST-Pi). Three model systems were considered for simulations in the protein environments the free enzyme, the complex between glutathione and the enzyme, and the complex between 1-chloro-2.4-dinitrobenzene, glutathione, and the enzyme. The structures derived from Molecular Dynamics simulations were compared with the crystallographic data available for the complex between the inhibitor S-(p-nitrobenzyl)glutathione and GST-Pi, the glutathione-bound form of GST-Pi, and the free enzyme carboxymethylated in Cys47. Free-energy perturbation techniques were used to determine the thermodynamics quantities for ionization of the phenol and thiol groups. The functional implications of Tyr7 in the activation of the glutathione thiol group are discussed in the light of present results, which in agreement with previous studies suggest that Tyr7 in un-ionized form contributes to the catalytic process of glutathione S-transferase, the thiolate anion being stabilized by hydrogen bond with Tyr7 and by interactions with hydrating water molecules.
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PMID:On the reaction mechanism of class Pi glutathione S-transferase. 926 69

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo-p-dioxin. The first step of the pathway is performed by a ring-dihydroxylating enzyme. Bunz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bunz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467-6475]. The gene encoding this [2Fe-2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested-PCR amplification. Primers for the amplification were designed based on the N-terminus sequence of the purified ferredoxin and on sequence comparisons with related proteins. Several cosmids were obtained and the ferredoxin gene, fdx1, was subcloned from one of them. The nucleotide sequence of a 4.6-kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi-component dioxygenases, genes encoding the alpha and beta subunits are found to be contiguous with the gene of the specific electron carrier, the fdx1 gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylases/isomerases and a glutathione S-transferase. The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin-type [2Fe-2S] cluster in this protein. Its redox potential was determined to be -245 (+/- 5) mV versus the normal hydrogen electrode at 25 degrees C, pH 8.0. Therefore, the protein is closely related to [2Fe-2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin-type [2Fe-2S] ferredoxin, namely Fdx1, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.
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PMID:Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1. 928 5

The filamentous fungus Penicillium chrysogenum showed remarkable resistance to the oxidative stress caused by high concentrations of either hydrogen peroxide (0.35-0.70 M) or tert-butyl hydroperoxide (tert-BOOH, 0.5-2.0 mM), which could be explained well with high levels of glutathione (GSH) peroxidase and catalase activities. The majority of exogenous H2O2 was likely removed by catalase from the cells while tert-BOOH was likely eliminated mainly by the GSH-dependent pathways. The GSH pool decreased considerably at high tert-BOOH concentrations, the glutathione disulphide (GSSG) pool increased at high H2O2 and tert-BOOH concentrations, meanwhile all the peroxide concentrations tested increased markedly the intracellular peroxide concentration. All the enzyme activities taking part in the glutathione metabolism (glutathione peroxidase, glutathione reductase, gamma-glutamyltranspeptidase and glutathione producing activities) except glutathione S-transferase increased significantly after exposing mycelia to both peroxides while the specific glucose-6-phosphate dehydrogenase and catalase activities remained unchanged. In the presence of 0.5 mM diamide both GSSG and GSH concentrations as well as the glutathione reductase and glutathione producing activities were elevated but no significant changes were found in the intracellular peroxide concentration or in any of the other enzyme activities examined.
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PMID:Glutathione metabolism and protection against oxidative stress caused by peroxides in Penicillium chrysogenum. 929 59

The 1241-bp promoter region of the Schistosoma mansoni 28-kDa glutathione S-transferase gene (Sm28GST) was sequentially deleted and analyzed using the luciferase reporter gene system in different cell lines. The activator protein-1 (AP-1) site located at -231 seems to be responsible for the major part of the promoter activity. The 1241-bp Sm28GST promoter was not, in transient transfection experiments, activated by reagents generating reactive oxygen species, such as hydrogen peroxide (H2O2), 3-methylcholanthrene, and ter-methylhydroquinone, but was significantly stimulated by phorbol 12-myristate 13-acetate, a potent protein kinase C activator. The involvement of the -231 AP-1 site in phorbol 12-myristate 13-acetate stimulation was demonstrated. Moreover, evidence for in vitro and in vivo binding of the -231 AP-1 site to Jun/Fos dimers was obtained using mobility gel shift assays and co-transfection of embryonic F9 cells with Jun/Fos expression plasmids, respectively. The presence in S. mansoni nuclear extracts of components with affinity for the AP-1 site suggests conservation of this regulatory pathway in the parasite.
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PMID:Deletion analysis of the Schistosoma mansoni 28-kDa glutathione S-transferase gene promoter in mammalian cells--importance of a proximal activator-protein-1 site. 931 Mar 68


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