Affinity labeling of pig lung glutathione S-transferase pi by 4-(fluorosulfonyl)benzoic acid

The compound 4-(fluorosulfonyl)benzoic acid (4-FSB) functions as an affinity label of the dimeric pig lung pi class glutathione S-transferase yielding a completely inactive enzyme. Protection against inactivation is provided by glutathione-based ligands, suggesting that the reaction target is near or part of the glutathione binding site. Radioactive 4-FSB is incorporated to the extent of 1 mol per mole of enzyme subunit. Peptide mapping revealed that 4-FSB reacts with two tyrosine residues in the ratio 69% Tyr7 and 31% Tyr106. The ratio is not changed by the addition of ligands. The results suggest that only one of the tyrosine residues can be labeled in the active site of a given subunit; i.e., reactions with Tyr7 and Tyr106 are mutually exclusive. We propose that the difference in labeling of these tyrosine residues is related to their pKa values, with Tyr7 exhibiting the lower pKa. The modified enzyme no longer binds to a S-hexylglutathione-agarose affinity column, even when only one of the active sites contains 4-FSB; these results may reflect interaction between the subunits. We conclude that Tyr7 and Tyr106 of the pig lung class pi glutathione S-transferase are important for function and are located at or close to the substrate binding site of the enzyme.     

Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione.

In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.     

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.     

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.     

Identification of tyrosine 79 in the tocopherol binding site of glutathione S-transferase pi

Alpha-tocopherol, the most abundant form of vitamin E present in humans, is a noncompetitive inhibitor of glutathione S-transferase pi (GST pi), but its binding site had not been located. Tocopherol iodoacetate (TIA), a reactive analogue, produces a time-dependent inactivation of GST pi to a limit of 25% residual activity. The rate constant for inactivation, k(obs), exhibits a nonlinear dependence on reagent concentration, with K(I) = 19 microM and k(max) = 0.158 min(-)(1). Complete protection against inactivation is provided by tocopherol and tocopherol acetate, whereas glutathione derivatives, electrophilic substrate analogues, buffers, or nonsubstrate hydrophobic ligands have little effect on k(obs). These results indicate that TIA reacts as an affinity label of a distinguishable tocopherol binding site. Loss of activity occurs concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. Isolation of the labeled peptide from the tryptic digest shows that Tyr(79) is the only enzymic amino acid modified. The Y79F, Y79S, and Y79A mutant enzymes were generated, expressed, and purified. Changing Tyr(79) to Ser or Ala, but not Phe, renders the enzyme insensitive to inhibition by either tocopherol or tocopherol acetate as demonstrated by increases of at least 49-fold in K(I) values as compared to the wild-type enzyme. These results and examination of the crystal structure of GST pi suggest that tocopherols bind at a novel site, where an aromatic residue at position 79 is essential for binding.     

The three-dimensional structure of class pi glutathione S-transferase in complex with glutathione sulfonate at 2.3 A resolution

The three-dimensional structure of class pi glutathione S-transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1-74) consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81-207) contains five alpha-helices. The dimeric molecule is globular with dimensions of about 55 A x 52 A x 45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S-transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.     

Tyrosine-7 in human class Pi glutathione S-transferase is important for lowering the pKa of the thiol group of glutathione in the enzyme-glutathione complex.

Previously, we reported the importance of Tyr7 for the catalytic activity of human class Pi glutathione S-transferase [Kong et al. (1992) Biochem. Biophys. Res. Comm., 182, 1122]. As an extension of this study, we investigated the pH dependence of kinetic parameters of the wild-type enzyme and the Y7F mutant. The replacement of Tyr7 with phenylalanine was found to alter the pH dependence of Vmax and Vmax/KmCDNB of the enzyme for conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB). The pKa of the thiol of GSH in the wild-type enzyme-GSH complex was estimated to be about 2.4 pK units lower than that in the Y7F-GSH complex. Tyr7 is thus considered to be important for catalytic activity in lowering the pKa of the thiol of GSH in the enzyme-GSH complex.     

Functional role of the lock and key motif at the subunit interface of glutathione transferase p1-1

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr(50) in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr(50) to Ala. Although the key residue is located far from the catalytic center, the k(cat) value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the k(cat) value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr(50), thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix alpha2, which interacts directly with GSH.     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase

Human class Alpha glutathione transferase (GST) A1-1 has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Tyr8----Phe lowers the specific activities with three substrates to 2-8% of the values for the wild-type enzyme. The changes in the kinetic parameters kcat/KM, Vmax and S0.5 show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered kcat values, suggesting that the hydroxyl group of Tyr8 is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (1991) EMBO J. 10, 1997-2005]. Thus, Tyr8 appears to be the first active site residue established as participating in the chemical mechanism of a GST.     

Mapping the substrate-binding site of a human class mu glutathione transferase using nuclear magnetic resonance spectroscopy.

The substrate-binding site of a human muscle class mu glutathione transferase has been characterized using high-resolution nuclear magnetic resonance spectroscopy. Isotopic labeling has been used to simplify one-dimensional proton NMR spectra of the Tyr and His residues in the enzyme and two-dimensional carbon-proton spectra of the Ala and Met residues in the enzyme. The resonance lines from 8 of the 12 Tyr residues have been assigned using site-directed mutagenesis. Replacement of Tyr7 with Phe reduced the activity of the enzyme 100-fold. The proximity of His, Tyr, Ala, and Met residues to the active site has been determined using a nitroxide-labeled substrate analogue. This substrate analogue binds with high affinity (Keq = 10(6) M-1) to the enzyme and is a competitive inhibitor. None of the His residues are within 17 A of the active site. Three of the assigned Tyr residues are greater than 17 A from the active site. Quantitative measurement of paramagnetic line broadening of five additional Tyr residues places them within 13-17 A from the active site. Broadening of the Ala and Met resonance lines by the spin-labeled substrate indicates that three Ala residues are 9-16 A from the nitroxide, three Met residues are less than 9 A from the nitroxide, and two Met residues are 9-16 A from the nitroxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.     

On the reaction mechanism of class Pi glutathione S-transferase

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 environment: 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. Proteins 28:530–542, 1997 © 1997 Wiley-Liss, Inc.     

Specific inactivation of glutathione S-transferases in class Pi by SH-modifiers

Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.     

Amino acid sequence of the major form of toad liver glutathione transferase

To investigate structural relationship between amphibian and mammalian GSTs the complete amino acid sequence of the major form of glutathione transferase present in toad liver (Bufo bufo) was determined. The enzyme subunit is composed of 210 amino acid residues corresponding to a molecular mass of 24,178 Da. In comparison with the primary structure of amphibian bbGSTP1-1, toad liver GST showed 54% sequence identity. On the other hand, toad liver GST showed about 45-55% sequence identity when compared with other pi class GST and less then 25% identity with GST of other classes. Amino acid residues involved in the H site and in the key and lock structure of the toad enzyme are significantly different from those of bbGSTP1-1 and other mammalian pi class GST. On the basis of its structural and immunological properties the toad liver GST, indicated as bbGSTP2-2, could represent the prototype of a subset of the pi family.     

Catalytically active monomer of glutathione S-transferase pi and key residues involved in the electrostatic interaction between subunits

Human glutathione transferase pi (GST pi) has been crystallized as a homodimer, with a subunit molecular mass of approximately 23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST pi, resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K(m) for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K(m)(GSH), whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V(max). The GST pi retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.     

An asparagine-phenylalanine substitution accounts for catalytic differences between hGSTM3-3 and other human class mu glutathione S-transferases

The hGSTM3 subunit, which is preferentially expressed in germ-line cells, has the greatest sequence divergence among the human mu class glutathione S-transferases. To determine a structural basis for the catalytic differences between hGSTM3-3 and other mu class enzymes, chimeric proteins were designed by modular interchange of the divergent C-terminal domains of hGSTM3 and hGSTM5 subunits. Replacement of 24 residues of the C-terminal segment of either subunit produced chimeric enzymes with catalytic properties that reflected those of the wild-type enzyme from which the C-terminus had been derived. Deletion of the tripeptide C-terminal extension found only in the hGSTM3 subunit had no effect on catalysis. The crystal structure determined for a ligand-free hGSTM3 subunit indicates that an Asn212 residue of the C-terminal domain is near a hydrophobic cluster of side chains formed in part by Ile13, Leu16, Leu114, Ile115, Tyr119, Ile211, and Trp218. Accordingly, a series of point mutations were introduced into the hGSTM3 subunit, and it was indeed determined that a Y119F mutation considerably enhanced the turnover rate of the enzyme for nucleophilic aromatic substitution reactions. A more striking effect was observed for a double mutant (Y119F/N212F) which had a k(cat)/K(m)(CDNB) value of 7.6 x 10(5) s(-)(1) M(-)(1) as compared to 4.9 x 10(3) s(-)(1) M(-)(1) for the wild-type hGSTM3-3 enzyme. The presence of a polar Asn212 in place of a Phe residue found in the cognate position of other mu class glutathione S-transferases, therefore, has a marked influence on catalysis by hGSTM3-3.     

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.