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At least 163 records · Page 9Linked to original sources
PubMed1998

The three-dimensional structure of Cys-47-modified mouse liver glutathione S-transferase P1-1. Carboxymethylation dramatically decreases the affinity for glutathione and is associated with a loss of electron density in the alphaB-310B region.

The three-dimensional structure of mouse liver glutathione S-transferase P1-1 carboxymethylated at Cys-47 and its complex with S-(p-nitrobenzyl)glutathione have been determined by x-ray diffraction analysis. The structure of the modified enzyme described here is the first structural report for a Pi class glutathione S-transferase with no glutathione, glutathione S-conjugate, or inhibitor bound. It shows that part of the active site area, which includes helix alphaB and helix 310B, is disordered. However, the environment of Tyr-7, an essential residue for the catalytic reaction, remains unchanged. The position of the sulfur atom of glutathione is occupied in the ligand-free enzyme by a water molecule that is at H-bond distance from Tyr-7. We do not find any structural evidence for a tyrosinate form, and therefore our results suggest that Tyr-7 is not acting as a general base abstracting the proton from the thiol group of glutathione. The binding of the inhibitor S-(p-nitrobenzyl)-glutathione to the carboxymethylated enzyme results in a partial restructuring of the disordered area. The modification of Cys-47 sterically hinders structural organization of this region, and although it does not prevent glutathione binding, it significantly reduces the affinity. A detailed kinetic study of the modified enzyme indicates that the carboxymethylation increases the Km for glutathione by 3 orders of magnitude, although the enzyme can function efficiently under saturating conditions.

Animals↗
PubMed1987

Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity.

The intracellular concentrations of total glutathione, GSSG and protein X S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione. A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms. This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis. A deficiency in catalase activity also produced an increase in the oxidation of glutathione. The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium. Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis.

Catalase↗
PubMed1990

Chemotactic activity for human PMN generated during ethanol metabolism by rat hepatocytes: role of glutathione and glutathione peroxidase.

Infiltration of the liver by polymorphonuclear leukocytes is a characteristic feature of alcoholic hepatitis. We have previously shown that liver cytosol metabolizing ethanol generates chemoattractant activity for polymorphonuclear leukocytes and that hydroxyl radical scavengers inhibit this process. To investigate the possibility that endogenous glutathione and glutathione peroxidase also inhibit this process, we evaluated chemoattractant activity production by glutathione and glutathione peroxidase deficient rat liver cytosol during ethanol metabolism. Incubation of cytosol deficient in both glutathione and glutathione peroxidase with 10 mM ethanol for 1 hour resulted in a 500-fold increase in chemoattractant activity when compared to cytosol with normal glutathione and glutathione peroxidase content. These findings provide further evidence for a role of oxygen free radicals in the generation of chemotactic activity and they also suggest that the ethanol-induced decrease in hepatic glutathione and glutathione peroxidase reported by others may have a significant potentiating effect on the recruitment of pro-inflammatory cells into the liver.

Animals↗
PubMed1997

Modulation of glutathione conjugation in vivo: how to decrease glutathione conjugation in vivo or in intact cellular systems in vitro.

Glutathione conjugation is involved in detoxification and toxification of a variety of electrophilic substrates. Thus it plays a major role in protection against reactive intermediates. At the same time this conjugation may cause resistance of tumor cells against certain cytostatics. In this review the methods available to decrease glutathione conjugation in vivo are discussed. So far the only in vivo active inhibitors of glutathione S-transferases are ethacrynic acid and a number of glutathione-derived structures; the latter seem very promising for further development. For (chronic) glutathione-depletion, buthionine sulfoximine is most effective, and surprisingly safe in clinical studies. Diethylmaleate can be used for acute depletion. Inhibition of glutathione transferases offers advantages over glutathione depletion as a method of decreasing glutathione conjugation since inhibition may be accomplished without changing the activities of other glutathione-dependent reactions in the cell. However, clinically safe, in vivo effective and isoenzyme-selective glutathione S-transferase inhibitors have not yet been developed.

Animals↗
PubMed1999

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man.

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.

Animals↗
PubMed1997

The three-dimensional structure of the human Pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate.

The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in chemotherapy. Its target is the detoxifying enzyme glutathione transferase which is often found overexpressed in cancer tissues. We have solved the crystal structures of human pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Ethacrynic acid is found to bind in a nonproductive mode to one of the ligand binding sites of the enzyme (the H site) while the glutathione binding site (G site) is occupied by solvent molecules. There are no structural rearrangements of the G site in the absence of ligand. The structure indicates that bound glutathione is required for ethacrynic acid to dock into the H site in a productive binding mode. The binding of the ethacrynic acid-glutathione conjugate shows that the contacts of the glutathione moiety with the protein are identical to those observed in crystal structures of the enzyme with other glutathione-based substrates and inhibitors. The ethacrynic acid moiety of the conjugate binds in the H site in a fashion that has not been observed in crystal structures of other glutathione-based inhibitor complexes. The crystal structures implicate Tyr 108 as an electrophilic participant in the Michael addition of glutathione to ethacrynic acid.

Crystallography, X-Ray↗
PubMed1993

Effects of the phenacetin metabolite 4-nitrosophenetol on the glutathione status and the transport of glutathione S-conjugates in human red cells.

The extent of ferrihemoglobin formation in human erythrocytes by 4-nitrosophenetol and its metabolisation rate strongly depended on the availability of cellular GSH. Ferrihemoglobin formation rate was increased by inhibition of the red cell glutathione reductase, and 4-nitrosophenetol disappeared more slowly. When red cells were completely depleted from SH groups, ferrihemoglobin formation was retarded, despite 4-nitrosophenetol was hardly metabolized. In turn, the glutathione status of human red cells was strongly affected by 4-nitrosophenetol. GSSG, which was produced in large amounts, was reduced, as long as the reducing system was intact. The decreased total glutathione content, however, did not recover completely, indicating formation of stable glutathione S-conjugates. The active export of the stable model glutathione thioether S-(2,4-dinitrophenyl)glutathione was strongly inhibited by 4-nitrosophenetol. A Lineweaver-Burk plot of the transport data suggested a competitive inhibition mechanism, presumably caused by glutathione adducts. The results indicate that the strong pi-donor substituent in 4-nitrosophenetol enables metabolic reactions with glutathione, producing biological effects hitherto not observed with nitrosobenzene. Bicyclic arylamines and glutathione S-conjugates may cause ferrihemoglobin formation that is not brought about by the diaphorase reaction. The latter may be responsible for transport inhibition of GSSG and other glutathione S-conjugates.

Biological Transport↗
PubMed1990

Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. Detection of the glutathione disulfide radical anion and the ascorbyl radical.

The acetaminophen phenoxyl radical was generated by the oxidation of acetaminophen by horseradish peroxidase in a fast-flow ESR experiment, and its reaction with glutathione and ascorbate was studied. Glutathione reduces the phenoxyl radical of acetaminophen to regenerate acetaminophen and form the thiyl radical of glutathione. This thiyl radical reacts with the thiolate anion of glutathione to form the disulfide radical anion, which was detected and characterized by ESR spectroscopy. In the presence of ascorbate, the ascorbyl radical was produced by the reduction of the acetaminophen phenoxyl radical by ascorbate. This reaction results in the complete reduction of the free radical of acetaminophen, whereas the glutathione reduction of the phenoxyl radical of acetaminophen was not complete on the fast-flow ESR time scale of milliseconds. This suggests that ascorbate rather than glutathione is more likely to react with the acetaminophen phenoxyl free radical in vivo. In the presence of both ascorbate and higher concentrations of glutathione, the reaction with ascorbate is dominant. When cysteine was used in the place of reduced glutathione in the above assay system, the disulfide radical anion of cystine was observed in a manner similar to glutathione. These reactions may have significance in the detoxification of acetaminophen and the free radical metabolites of xenobiotics in general. Only in cells containing low levels of ascorbate can glutathione play a direct role in the detoxification of the acetaminophen phenoxyl radical.

Acetaminophen↗
PubMed2002

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver.

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.

Animals↗
PubMed1990

Glutathione synthesis and glutathione redox pathways in naphthalene cataract of the rat.

This investigation examined many parameters during the course of early development of naphthalene-induced cataract in a time span of 0 to 79 days of treatment. Feeding naphthalene daily to Black-Hooded rats resulted in gradual progressive development of cataract. The first faint opacities were detectable after 7 days. Free soluble total glutathione (oxidized and reduced) of these lenses was shown to gradually decrease to a maximum loss of about 20%, a value reached by day 30 of treatment. No activity loss of either enzyme required for glutathione synthesis (gamma-glutamylcysteine synthetase or glutathione synthetase) was observed in homogenates of naphthalene versus control lenses. There was also neither impairment of [35S]-L-cystine uptake nor of [35S]-glutathione synthetic capacity in lenses cultured from rats after 12, 24 or 36 days of naphthalene feeding when compared to control lenses. Hence, glutathione loss cannot be explained by a damaged glutathione synthesis system. Progressive activity loss of glutathione peroxidase and glutathione reductase was observed. The loss of glutathione peroxidase activity was especially remarkable. Thus, the defense system against oxidative damage is impaired and may be a significant factor in naphthalene-induced cataract of the rat.

Animals↗
PubMed1986

Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase.

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.

Binding Sites↗
PubMed1990

Growth state dependent increase of glutathione by homocysteine and other thiols, and homocysteine formation in glutathione depleted mouse cell lines.

Homocysteine has been shown to increase glutathione levels in C3H/10T1/2 Cl 8 cells. The present paper confirms that this increase was specific for non-dividing cells. Several other thiols and disulfides, including cysteamine, mercaptoethanol and dithioerythritol, also increased glutathione, but the specificity for quiescent non-dividing cells was confined to homocysteine only. Cysteamine was most efficient, increasing glutathione 5-fold in confluent, non-dividing cells, and 3.2-fold in exponentially growing Cl 8 cells. The results indicate that the increase in glutathione was not specific for homocysteine or other cysteine generating agents, but rather related to the presence of potential thiol, either in free form, as thiolactone or in its oxidized, disulfide form. The effect of the glutathione synthesis inhibitor BSO was investigated in detail in both C3H/10T1/2 Cl 8 cells and in R1.1. mouse lymphoma cells. Twenty-four hours after addition of 20 microM BSO to exponential growing Cl 8 cells the glutathione content was reduced to 5.5%, with minimal toxic effect. To achieve the same GSH depleting effect on exponential growing R1.1. cells, the BSO concentration had to be increased to 50 microM, which had a slight, but distinct growth inhibitory effect on the lymphoma cells. Based on these data, the possibility that glutathione mediated homocysteine production was investigated in part by depleting the cells of glutathione and determining the homocysteine export rate as a measure of the intracellular production of the metabolite. The results showed that glutathione depletion by BSO had no effect on the homocysteine export rate in Cl 8 cells, while in R1.1. cells a moderate decrease in homocysteine export rate accompanied by a slight, but distinct decrease in growth rate, was observed when the cells were depleted of glutathione. In addition, these data indicate that BSO did not interfere with the overall transmethylation rate, and this observation supports the view of BSO as a specific inhibitor of GSH synthesis. A general difference between the homocysteine export rate in Cl 8 and R1.1. cells was observed. The former demonstrated a decreasing export rate during exponential growth, while the latter showed an initial decrease and then a slight increase in homocysteine export rate.

Animals↗
PubMed1981

Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation.

Glutathione (in the form of GSH) is transported out of cultured human lymphoid cells at rates proportional to the intracellular glutathione levels. Inhibition of glutathione synthesis by buthionine sulfoximine, a potent selective inhibitor of gamma-glutamylcysteine synthetase, leads to exponential decrease in intracellular glutathione, a large fraction of which appears extracellularly, indicating that glutathione turnover is associated with its export. Although cells with 0.09 mM glutathione (4% of controls) were 85% viable, further decrease was associated with marked loss of viability. Cells with 4-5% of control glutathione levels were much more sensitive than control cells to the effects of gamma radiation and of 5,5'-dithiobis(2-nitrobenzoate). Depletion of glutathione by use of buthionine sulfoximine has advantages over other reagents (such as diamide, other oxidizing agents, and diethylmaleate, which affect other cellular components and may increase glutathione disulfide levels) and therefore has potential usefulness in sensitizing cells to the effects of radiation and to therapeutic agents that are detoxified by reactions involving glutathione.

Biological Transport↗
PubMed2001

The biological functions of glutathione revisited in arabidopsis transgenic plants with altered glutathione levels.

A functional analysis of the role of glutathione in protecting plants from environmental stress was undertaken by studying Arabidopsis that had been genetically modified to have altered glutathione levels. The steady-state glutathione concentration in Arabidopsis plants was modified by expressing the cDNA for gamma-glutamyl-cysteine synthetase (GSH1) in both the sense and antisense orientation. The resulting plants had glutathione levels that ranged between 3% and 200% of the level in wild-type plants. Arabidopsis plants with low glutathione levels were hypersensitive to Cd due to the limited capacity of these plants to make phytochelatins. Plants with the lowest levels of reduced glutathione (10% of wild type) were sensitive to as little as 5 microM Cd, whereas those with 50% wild-type levels required higher Cd concentrations to inhibit growth. Elevating glutathione levels did not increase metal resistance. It is interesting that the plants with low glutathione levels were also less able to accumulate anthocyanins supporting a role for glutathione S-transferases for anthocyanin formation or for the vacuolar localization and therefore accumulation of these compounds. Plants with less than 5% of wild-type glutathione levels were smaller and more sensitive to environmental stress but otherwise grew normally.

Anthocyanins↗
PubMed1987

Renal clearance of glutathione measured in rats pretreated with inhibitors of glutathione metabolism.

Renal clearance measurements were performed in rats pretreated with various inhibitors of glutathione metabolism. Approximately 97% of the renal gamma-glutamyltranspeptidase was inactivated by infusion of AT-125. Within 4.5 h arterial plasma concentration and urinary excretion of glutathione increased from 4 microM and 1.2 nmol/h to 27 microM and 3,900 nmol/h, respectively. The ratio of excreted to filtered glutathione increased from less than 0.01 to 1. When renal glutathione was decreased to 35% of normal by pretreating rats with D,L-buthionine-S,R-sulfoximine, the subsequent inactivation of the transpeptidase caused only a two-fold increase in arterial plasma glutathione, and urinary excretion increased to only 70% of the filtered load. The filtered load of glutathione was reduced by intravenous infusion of purified gamma-glutamyltranspeptidase. At 1 to 3 h after infusion, arterial glutathione decreased to 0.3 microM. Under these conditions, the amount of glutathione excreted by an AT-125-treated rat was 40-fold greater than the filtered load. When similar experiments were carried out in rats that were pretreated with D,L-buthionine-S,R-sulfoximine and AT-125, urinary excretion of glutathione was decreased 10-fold, but it still exceeded the amount filtered. These results firmly establish that apical secretion contributes to the glutathione catabolized in the tubular lumen.

Animals↗
PubMed1991

Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions.

Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO(2) assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway.

Journal Article↗
PubMed1996

Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture.

Glutathione S-transferases (GST) are a family of multifunctional enzymes involved in the metabolization of a broad variety of xenobiotics and reactive endogenous compounds. The interest in plant glutathione S-transferases may be attributed to their agronomic value, since it has been demonstrated that glutathione conjugation for a variety of herbicides is the major resistance and selectivity factor in plants. The three-dimensional structure of glutathione S-transferase from the plant Arabidopsis thaliana has been solved by multiple isomorphous replacement and multiwavelength anomalous dispersion techniques at 3 A resolution and refined to a final crystallographic R-factor of 17.5% using data from 8 to 2.2 A resolution. The enzyme forms a dimer of two identical subunits each consisting of 211 residues. Each subunit is characterized by the GST-typical modular structure with two spatially distinct domains. Domain I consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side by an irregular segment containing three short 3(10)-helices, while domain II is entirely helical. The dimeric molecule is globular with a prominent large cavity formed between the two subunits. The active site is located in a cleft situated between domains I and II and each subunit binds two molecules of a competitive inhibitor S-hexylglutathione. Both hexyl moieties are oriented parallel and fill the H-subsite of the enzyme's active site. The glutathione peptide of one inhibitor, termed productive binding, occupies the G-subsite with multiple interactions similar to those observed for other glutathione S-transferases, while the glutathione backbone of the second inhibitor, termed unproductive binding, exhibits only weak interactions mediated by two polar contacts. A most striking difference from the mammalian glutathione S-transferases, which share a conserved catalytic tyrosine residue, is the lack of this tyrosine in the active site of the plant glutathione S-transferase.

Amino Acid Sequence↗
PubMed1998

Three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate: catalytic roles of Cys10 and His106.

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.

Amino Acid Sequence↗