Site-directed mutagenesis of glutathione S-transferase YaYa: nonessential role of histidine in catalysis

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.     

The glutathione S-transferase activity in the kidney of rainbow trout (Salmo gairdneri)

Trout kidney contains 2.3 mmol GSH/kg. The cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.35 mumol/min/mg protein. There is no detectable activity with 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, p-nitrobenzyl chloride or p-nitrophenyl acetate. A variable proportion of the activity does not bind to a glutathione-affinity matrix. Its Km values for GSH and 1-chloro-2,4-dinitrobenzene are 3.0 and 5.1 mM, respectively. The rest of the activity is eluted from the affinity matrix as one main and two minor peaks. The main peak has Km values for GSH and 1-chloro-2,4-dinitrobenzene of 0.4 and 4.5 mM, respectively. Its subunit Mr is 22,900. The activity in the main peak is inhibited progressively by 1-chloro-2,4-dinitrobenzene with a rate constant of 0.11/min.     

Distinctions between the multiple cationic forms of rat liver glutathione S-transferase

Three cationic glutathione S-transferase forms isolated from rat liver were characterized as dimers that originated from different combinations of two subunit types, Ya and Yc. The cationic forms were purified using lysyl glutathione affinity matrices and were chromatographically resolved from anionic glutathione S-transferases that contain Yb subunits. The three classes of cationic transferase exhibited similar specific activities with 1-chloro-2,4-dinitrobenzene as a substrate, all forms cross-reacted with antibodies to glutathione S-transferase B, and all had comparable secondary structures and tryptophan fluorescence properties. In spite of those similarities, the Yc-containing forms were clearly distinguishable from Ya forms on the basis of characteristic differences in circular dichroic patterns associated with their aromatic side chains. All cationic transferases bound bilirubin with stoichiometric ratios of 1 mol/dimeric protein molecule, but discrete differences in mode of binding were ascribed to forms containing Ya subunits as compared to Yc dimers. Binding to Yc forms was of lower affinity and may be associated with the catalytic region of the protein since glutathione effectively displaced bilirubin from the Yc component.     

Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase.

In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T. et al. (1987) Cancer Res., 47, 5626-5630) and an E. coli expression system. The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid. On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14. These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.     

The glutathione S-transferase activity in the pyloric caeca of rainbow trout, Salmo gairdneri

Pyloric caeca of trout contain 1.9 mmol GSH/kg tissue. Cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.06 mmol/min/g protein. Cholate (3.3 mM) inhibits cytosolic transferase activity by 55% at pH 6.6 and by 4% at pH 7.4. The transferases do not bind 8-anilino-1-naphthalene sulphonate at pH 7.4. The cytosolic transferases are inactivated progressively by 1-chloro-2,4-dinitrobenzene, 50% of their activity being lost in 5.0 min. A minority of the activity does not bind to a glutathione-affinity matrix. At pH 6.6 its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.88 and 9.1 mM respectively. The rest of the activity is eluted from the affinity matrix as a single peak. Its apparent Michaelis constants for GSH and 1-chloro-2,4-dinitrobenzene are 0.33 and 2.9 mM respectively. Its subunit Mr is 22.4 kDa.     

Glutathione S-transferases of the bovine retina. Evidence that glutathione peroxidase activity is the result of glutathione S-transferase.

We have purified two isoenzymes of glutathione S-transferase from bovine retina to apparent homogeneity through a combination of gel-filtration chromatography, affinity chromatography and isoelectric focusing. The more anionic (pI = 6.34) and less anionic (pI = 6.87) isoenzymes were comparable with respect to kinetic and structural parameters. The Km for both substrates, reduced glutathione and 1-chloro-2,4-dinitrobenzene, bilirubin inhibition of glutathione conjugation to 1-chloro-2,4-dinitrobenzene, 1-chloro-2,4-dinitrobenzene inactivation of enzyme activity and molecular weight were similar. However, pH optimum and energy of activation were found to differ considerably. Retina was found to have no selenium-dependent glutathione peroxidase activity. The total glutathione peroxidase activity fractionated with the transferases in the gel-filtration range of mol.wt. 49000 and expressed activity with only organic hydroperoxides as substrate. Only the more anionic isoenzyme expressed both transferase and peroxidase activity.     

Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition

The procedure developed for purification of the N-ethylmaleimide-activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition. In addition the microsomal glutathione transferase purified in unactivated form could be activated 15-fold with N-ethylmaleimide to give the same specific activity with 1-chloro-2,4-dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N-ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'-dithiopyridyl and binding of radioactive N-ethylmaleimide. Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1. Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N-ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.     

The purification and characterization of glutathione S-transferase from the hepatopancreas of the blue crab, Callinectes sapidus

High glutathione S-transferase activity was found in the cytosol of F-cells from the hepatopancreas of the blue crab (Callinectes sapidus). Purification of glutathione S-transferase from hepatopancreas extracts by Sephadex G-200, DEAE-Sephacel, and chromatofocusing resulted in the isolation of two isozymes with isoelectric points of 5.9 and 5.7, as determined by analytical isoelectric focusing. Using 1-chloro-2,4-dinitrobenzene as the substrate the specific activities of the two purified isozymes were 222 and 182 mumol/min/mg, respectively. There was no evidence for basic transferase isozymes. In addition to 1-chloro-2,4-dinitrobenzene the purified glutathione S-transferase isozymes showed activity with p-nitrophenyl acetate, p-nitrobenzyl chloride, bromosulfophthalein, and benzopyrene oxide. Thus, both substitution and addition reactions associated with vertebrate glutathione S-transferase were found in the crab transferases. There was no when ethacrynic acid, methyl iodide, trans-4-phenyl-3-buten-2-one, 1,2-epoxy-(p-nitrophenoxy)propane, cumene hydroperoxide, and t-butyl hydroperoxide were used as substrates. The lack of peroxidase activity is of interest since this activity is commonly found in vertebrate transferase isozymes. The two transferases had a dimeric Mr of 40,800 with similar amino acid compositions and similar kinetic parameters (Vmax, Km, and pH maxima) with 1-chloro-2,4-dinitrobenzene as substrate. The two transferases could be distinguished by their isoelectric points, molecular mass of the monomers (22,300 for GST 1 and 22,300 and 22,400 for GST 2), and different inhibitor mechanisms with hematin and bromosulfophthalein.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as YaYa and YcYc, were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[3H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase YaYa toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the YaYa isozyme, whereas two labeled peptides were present in the tryptic digest of YcYc. The Ya peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Yc were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Ya peptide and the smaller peptide purified from Yc came from the same region of the Ya and Yc subunits. This common region and a second region of the Yc subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.     

Purification and some properties of glutathione S-transferase from Escherichia coli B.

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (Km, 1.43 mM) and glutathione (Km, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3,2 mumol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.     

Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica

A highly active glutathione S-transferase was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24 kDa by SDS/PAGE, and had a different electrophoretic mobility than, a previously identified Sigma class glutathione S-transferase (Bla g 5). Kinetic study of 1-chloro-2,4-dinitrobenzene conjugation revealed a high catalytic rate but common substrate-binding and cosubstrate-binding affinities, with V(max), k(cat), K(m) for 1-chloro-2,4-dinitrobenzene and K(m) for glutathione estimated to be 664 micromol x mg(-1) x min(-1), 545 s(-1), 0.33 mm and 0.76 mm, respectively. Interestingly, this enzyme possessed the highest activity for cumene hydroperoxide among insect glutathione S-transferases reported to date. Along with the ability to metabolize 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and 4-hydroxynonenal, this glutathione S-transferase may play a role in defense against insecticides as well as oxidative stress. On the basis of the amino acid sequences obtained from Edman degradation and MS analyses, a 987-nucleotide cDNA clone encoding a glutathione S-transferase (BggstD1) was isolated. The longest ORF encoded a 24 614 Da protein consisting of 216 amino acid residues. The sequence had close similarities ( approximately 45-60%) to that of Delta class glutathione S-transferases, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified glutathione S-transferase. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.     

Glutathione S-transferase composition of rat erythrocytes

With 1-chloro-2,4-dinitrobenzene as the electrophilic substrate, the specific activity of glutathione S-transferase in rat haemolysates was found to range from 0.002 to 0.013 mumol/min/mg haemoglobin at 30 degrees C. To establish the glutathione S-transferase composition, chromatofocusing was used which indicated the presence of a single soluble isoenzyme with an apparent pI of 6.1. A molecular weight of 48,000 was determined for the enzyme by gel filtration. The transferase enzyme in intact erythrocytes is shown to catalyze the formation of S-(2,4-dinitrophenyl)-glutathione from 1-chloro-2,4-dinitrobenzene and endogenous glutathione. Efflux of this conjugate from erythrocytes proceeded at a rate of 13 nmol/min/ml at 37 degrees C.     

Glutathione peroxidase activities from rat liver

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.     

Glutathione S-transferase isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M(r)s of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

Quantitative and qualitative gender-related differences in jejunal glutathione S-transferase in the rat effect of testosterone administration

Gender-related differences and the regulation by testosterone of glutathione S-transferase were studied in rat jejunum. We analyzed enzyme activity and the relative content of GST subunits. Four experimental groups of adult rats were studied: normal males, castrated males, castrated males injected with testosterone and normal females. Glutathione S-transferase activity was assayed using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Differences in subunit composition among groups were evaluated by western blot analysis. The results demonstrated that 1-chloro-2,4-dinitrobenzene conjugation rate is higher in normal males than in normal females and castrated males. Testosterone administration to castrated males raised the activity up to the level observed in normal males. No significant difference in glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was observed among groups. Western blot analysis revealed that males and females differ in all subunits tested that is, rGSTA2, rGSTM1, rGSTM2 and rGSTP1, and that testosterone regulates the content of rGSTM1, rGSTM2 and rGSTP1. In conclusion, jejunal GST shows a gender-dependent regulation affecting both enzyme activity and subunit composition, and testosterone appears to be one of the factors involved.     

Kinetic independence of the subunits of cytosolic glutathione transferase from the rat.

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.     

Anionic and neutral glutathione S-transferase isoenzymes in the freshwater worm Tubifex tubifex (O.F.M.)

The anionic and the neutral glutathione S-transferase (GST) isoenzymes from the freshwater worm Tubifex tubifex (O.F.M.) were separated in one step by chromatofocusing on a Polybuffer exchanger 94 column, eluted with Polybuffer 74. Their pI values ranged between 4.12 and 6.98, and their molecular weight between 30 000 and 38 400. The apparent Km values towards glutathione and 1-chloro-2,4-dinitrobenzene were also determined. The high number of non-cationic GST isoenzymes is unusual. Tubifex worms seems well equipped for attacking environmental pollutants.     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.