Human Placental Glutathione S-transferase Research Articles

Mechanistic insights into the inhibition of human placental glutathione S-transferase P1-1 by abscisic and gibberellic acids: An integrated experimental and computational study.

The interactions of the classic phytohormones gibberellic acid (gibberellin A3 , GA3 ) and abscisic acid (dormin, ABA), which antagonistically regulate several developmental processes and stress responses in higher plants, with human placental glutathione S-transferase P1-1 (hpGSTP1-1), an enzyme that plays a role in endo- or xenobiotic detoxification and regulation of cell survival and apoptosis, were investigated. The inhibitory potencies of ABA and GA3 against hpGSTP1, as well as the types of inhibition and the kinetic parameters, were determined by making use of both enzyme kinetic graphs and SPSS nonlinear regression models. The structural basis for the interaction between hpGSTP1-1 and phytohormones was predicted with the aid of molecular docking simulations. The IC50 values of ABA and GA3 were 5.3 and 5.0 mM, respectively. Both phytohormones inhibited hpGSTP1-1 in competitive manner with respect to the cosubstrates GSH and CDNB. When ABA was the inhibitor at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v , Vm , Km , and Ki values were statistically estimated to be 205 ± 16 μmol/min-mg protein, 1.32 ± 0.18 mM, 1.95 ± 0.25 mM and 175 ± 6 μmol/min-mg protein, 0.85 ± 0.06 mM, 1.85 ± 0.16 mM, respectively. On the other hand, the kinetic parameters Vm , Km , and Ki obtained with GA3 at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v were found to be 303 ± 14 μmol/min-mg protein, 1.77 ± 0.13 mM, 3.38 ± 0.26 mM and 249 ± 7 μmol/min-mg protein, 1.43 ± 0.07 mM, 2.89 ± 0.19 mM, respectively. Both phytohormones had the potential to engage in hydrogen-bonding and electrostatic interactions with the key residues that line the G- and H-sites of the enzyme's catalytic center. Inhibitory actions of ABA/GA3 on hpGSTP1-1 may guide medicinal chemists through the structure-based design of novel antineoplastic agents. It should be noted, however, that the same interactions may also render fetuses vulnerable to the potentially toxic effects of xenobiotics and noxious endobiotics.

Assessment of the inhibitory activity of the pyrethroid pesticide deltamethrin against human placental glutathione transferase P1-1: A combined kinetic and docking study

Deltamethrin (DEL), which is a synthetic pyrethroid insecticide, has been used successfully all over the world to treat mosquito nets for the control of malaria. Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the conjugation of reduced glutathione (GSH) to a variety of xenobiotics and are normally recognized as detoxification enzymes. Here, we used a colorimetric assay based on the human placental GSTP1-1 (hpGSTP1-1)-catalyzed reaction between GSH and the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) as well as molecular docking to investigate the mechanistic and structural aspects of hpGSTP1-1 inhibition by DEL. We show that DEL is a potent, noncompetitive inhibitor of hpGSTP1-1 with an IC50 value of 6.1 μM and Ki values of 5.61 ± 0.32 μM and 7.96 ± 0.97 μM at fixed [CDNB]–varied [GSH] and fixed [GSH]–varied [CDNB], respectively. DEL appears to be accommodated well in an eccentric cavity located at the interface of the hpGSTP1-1 homodimer, presumably causing conformational changes to the enzyme’s substrate-binding sites such that the enzyme is no longer able to transform GSH and CDNB effectively. Correspondingly, considerable maternal exposure to and subsequent accumulation of DEL may interfere with the proper development of the vulnerable fetus, possibly increasing the risk of developing congenital defects.

Model Studies on Peroxidic Glutathione Transferase (GST) Inhibitors: C5‐Methylated 1,2,4‐Trioxanes with C6‐Acrylate Side Chains

AbstractThe 1,2‐hydroxy hydroperoxide 7 was obtained with high threo diastereoselectivity from the ester‐conjugated allylic alcohol 6 through a porphyrin‐sensitized singlet oxygen ene reaction. Compound 7 was used as substrate for peroxy(trans)acetalization with aldehydes, orthoesters, and acetals, catalyzed by boron and indium Lewis acids, as well as by PPTS as a Brønsted acid catalyst (for trioxane 10). The 1,2,4‐trioxane 8 was formed with high diasteroselectivity with all‐equatorially positioned substitutents at C‐3, C‐5, and C‐6. Additionally, perorthoester 9 was obtained by indium(III)‐catalyzed condensation of an orthoester with the hydroperoxide 7. The new compounds 8 and 9 exhibited moderate inhibition of human placental glutathione transferase (GST), comparable to that shown by the 5‐unsubstituted model trioxane 11.

Coniferyl Ferulate, a Strong Inhibitor of Glutathione S-Transferase Isolated from RadixAngelicae sinensis, Reverses Multidrug Resistance and Downregulates P-Glycoprotein

Glutathione S-transferase (GST) is the key enzyme in multidrug resistance (MDR) of tumour. Inhibition of the expression or activity of GST has emerged as a promising therapeutic strategy for the reversal of MDR. Coniferyl ferulate (CF), isolated from the root of Angelica sinensis (Oliv.) Diels (Radix Angelicae sinensis, RAS), showed strong inhibition of human placental GST. Its 50% inhibition concentration (IC50) was 0.3 μM, which was greater than a known GSTP1-1 inhibitor, ethacrynic acid (EA), using the established high-throughput screening model. Kinetic analysis and computational docking were used to examine the mechanism of GST inhibition by CF. Computational docking found that CF could be fully docked into the gorge of GSTP1-1. The further exploration of the mechanisms showed that CF was a reversible noncompetitive inhibitor with respect to GSH and CDNB, and it has much less cytotoxicity. Apoptosis and the expression of P-gp mRNA were evaluated in the MDR positive B-MD-C1 (ADR+/+) cell line to investigate the MDR reversal effect of CF. Moreover, CF showed strong apoptogenic activity and could markedly decrease the overexpressed P-gp. The results demonstrated that CF could inhibit GST activity in a concentration-dependent manner and showed a potential MDR reversal effect for antitumour adjuvant therapy.

Human placental glutathione S-transferase activity and polycyclic aromatic hydrocarbon DNA adducts as biomarkers for environmental oxidative stress in placentas from pregnant women living in radioactivity- and chemically-polluted regions

This study was designed to analyze the effect of environmental oxidative stress on human placental monooxygenases, glutathione S-transferase (GST) activity and polycyclic aromatic hydrocarbon (PAH)–DNA adducts in human term placentas from radioactivity-contaminated and chemically-polluted areas of the Ukraine and Belarus, and to compare these biomarkers to the newborn's general health status. Placental PAH–DNA adduct formation, GST activity, 7-ethoxycoumarin O-deethylase (ECOD) activity, and thiobarbituric reactive substances (TBARS), an index of lipid peroxidation, were measured in groups of women exposed to different levels of radioactivity and PAH pollution. The in vitro metabolism data, obtained from 143 human placental samples at term, were compared to indices of maternal and newborn health. The highest ECOD activity was recorded in placentas obtained from chemically-polluted areas and a radioactivity-contaminated area; the ECOD activity was 7-fold and 2-fold higher compared to the region considered to be “clean”. Newborns with the most compromised health status displayed the greatest down-regulation of GST activity (144–162 mU mg protein −1 vs. 258–395 mU mg protein −1), enhanced ECOD activity and the highest level of PAH–DNA adduct formation. The highest level of TBARS was observed in women exposed to the highest levels of radiation. The efficiency of placental detoxification negatively correlated with maternal age and the health status of the newborn. Environmental oxidative stress was related to an increase in anemia, threatened abortions, toxemia, fetal hypoxia, spontaneous abortions and fetal hypotrophy. Our data suggest that chemically- or radioactivity-induced oxidative stress enhance cytochrome P450-mediated enzymatic activities potentially resulting in increased formation of reactive metabolites. The activity of GSH-transferase is not enhanced. This imbalance in detoxification capacity can be measured as increased production of PAH–DNA adducts, decreased lipid peroxidation and compromised fetal health.

Phosphono analogues of glutathione as new inhibitors of glutathione S-transferases.

Phosphono-analogues of glutathione containing the O = P(OR)2 moiety in place of the cysteinyl residue CH2SH 1a-1d were prepared by solution phase peptide synthesis. Benzyl, benzyloxy-carbonyl, and tert-butyl protecting groups were used to mask the individual amino acid functional groups. The formation of peptide bonds was achieved by the usual peptide synthesis via activation of carboxylic functions with cyclohexylcarbodiimide and subsequent reaction with free amino groups. The thus obtained, fully-protected peptides were each purified by normal phase column chromatography. Deprotection was accomplished by hydrogenolysis and by treatment with HBr/acetic acid yielding the desired phosphonic acid diester 1a-1d. The inhibition of the glutathione conjugation of 1-chloro-2,4-dinitrobenzene by human placental glutathione S-transferase was studied by determining the IC50 values of the new glutathione analogues. The IC50 values were 291 microM, 139 microM, 64 microM, and 21 microM for the dimethyl, diethyl, diisopropyl, and di-n-butyl esters, respectively. The results clearly show that the formal substitution of the glutathione thiol function by phosphonic acid esters leads to a new class of glutathione S-transferase inhibitors. Further investigations directed at the question of whether or not these glutathione analogues are suitable for a modulation in chemotherapy are in progress.

Site-directed Mutagenesis of Human Glutathione Transferase P1-1: SPECTRAL, KINETIC, AND STRUCTURAL PROPERTIES OF Cys-47 AND Lys-54 MUTANTS

In the human placental glutathione transferase, Cys-47 possesses, at physiological pH values, a pK alpha value of 4.2 and may exist as an ion pair with the protonated epsilon-amino group of Lys-54. Using site-directed mutagenesis we investigate spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. The results shown indicate that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47. The contribution of Lys-54 to the activation of Cys-47 is assessed by the spectral properties of the K54A mutant enzyme. The induced cooperativity toward glutathione, as a consequence of mutation of Lys-54 to alanine, clearly parallels that observed for the Cys-47 mutant enzymes (see the preceding paper (Ricci, G., Lo Bello, M., Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W., and Federici, G. (1995) J. Biol. Chem. 270, 1243-1248) and points out the importance of this electrostatic interaction in shaping the correct spatial arrangement for the binding of glutathione and in anchoring the flexible helix alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity.

Increase in placental glutathione S-transferase in human oral epithelial dysplastic lesions and squamous cell carcinomas.

This study investigated the immunohistochemical expression of human placental glutathione S-transferase (GST-pi) in the epithelium of oral premalignant and malignant lesions. Epithelial lining of normal oral mucosa, hyperplastic lesions and oral epithelium exhibiting mild dysplasia showed weak to moderate GST-pi staining. Moderate epithelial dysplasia revealed an increased antibody content while severe dysplasia, carcinoma-in-situ (CIS) and squamous cell carcinoma (SCC) demonstrated markedly increased antibody binding. The GST-pi staining was evident mainly in the cytoplasm. Severe dysplasia, CIS and SCC were also characterized by areas of cells with intensive nuclear GST-pi staining. These findings support the hypothesis that GST-pi plays a role in human oral carcinogenesis and may be used as a tumor marker for human oral premalignant and malignant lesions.

Peculiar spectroscopic and kinetic properties of Cys-47 in human placental glutathione transferase. Evidence for an atypical thiolate ion pair near the active site.

Cys-47, the most reactive cysteine in the homodimeric glutathione transferase (EC 2.5.1.18) from human placenta (class Pi), displays peculiar acid base and spectroscopic properties. The thiolate form of this residue is characterized by a sharp UV absorption spectrum centered at 229 nm with an epsilon = 7,500 M-1 cm-1. The dependence of the apparent extinction coefficient on pH indicates that the sulfhydryl group of Cys-47 has a pKa value of 4.2. Moreover the dependence of the reactivity of Cys-47 toward bromopyruvate and iodoacetamide with pH resembles that found for the functional sulfhydryls of thiol proteases, which have very low pKa values and exist mainly as a mercaptide-imidazole ion pair. The apparent pKa value for Cys-47, calculated by this kinetic approach, is in good agreement with that determined spectroscopically. X-ray crystallographic data indicate that the protonated amino group of Lys-54, 4.9 A from the sulfur atom, is probably involved in the deprotonation of Cys-47. Calculation of the electrostatic potential on the sulfur atom of Cys-47 gives a theoretical pKa value of 3.5 for the sulfhydryl group. The simulated neutralization of Lys-54 shifts the pKa value of Cys-47 to a normal value of 9.5. These findings suggest that at physiological pH values, Cys-47 exists as the thiolate ion stabilized by an ion pair formation with the protonated amino group of Lys-54, and this probably accounts for its high reactivity.

Conformational States of Human Placental Glutathione Transferase as Probed by Limited Proteolysis

Limited proteolysis experiments have been carried out on human placental glutathione transferase in its different forms. The reduced enzyme, as well as the oxidized form and that inactivated with cystamine were all sensitive to 10 % (w/w) trypsin, under nondenaturing conditions. The proteolytic cleavage was accompanied by a concomitant loss of enzymatic activity. On the contrary, the presence of glutathione or glutathione conjugates strongly protected the reduced enzyme against inactivation and from the proteolytic attack. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only the peptide bond between Lys 44 and Ala 45 was cleaved. Since Lys 44 has been demonstrated to be involved in the glutathione binding, it is suggested that the region surrounding this amino acid residue (α B helix) could be more exposed to the solvent, in the absence of glutathione. Crystallographic data also indicated that this region is flexible, supporting the idea that it may be involved in the observed conformational change upon glutathione binding.

Inhibition of human term placental and fetal liver glutathione- S-transferases by fatty acids and fatty acid esters

Glutathione- S-transferase (GST) activity from human term placenta and human fetal liver towards 1 -chloro-2,4-dinitrobenzene as the second substrate was significantly inhibited by the saturated fatty acids, stearic (SA) and palmitic (PA) acids and fatty acid esters, ascorbyl stearate (Asc-S) and ascorbyl palmitate (Asc-P). The nature of inhibition of human placental GST was competitive towards CDNB with K i values of 3.1, 10.0, 13.5 and 18.5 μM for Asc-S, Asc-P, PA and SA, respectively. The inhibitory effect of Asc-S on human term placental GST was reversible. I 50 values for Asc-S, Asc-P, SA and PA were 15, 45, 83 and 78 μM, respectively, for partially purified human fetal liver GSTs and 21, 6, 88 and 117 μM. respectively, for partially pure rat liver GSTs. The evidence suggests that Asc-S, Asc-P, SA and PA are potent inhibitors especially of the pi-class of GST.

Chemical modification of human placental glutathione transferase by pyridoxal 5′-phosphate

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.

1-Chloro-2,4-dinitrobenzene-mediated irreversible inactivation of acidic glutathione S-transferases Inactivation mechanism—A saturation-type or simple second-order kinetic process?

A critical analysis of the inactivation kinetics exhibited by the acidic human glutathione S-transferase (GST) enzymes is presented. Data on the 1-chloro-2,4-dinitrobenzene (CDNB)-facilitated inactivation of human placental GST π have been utilized in conjunction with published inactivation data from the literature to answer the following two questions: (a) do the inactivation kinetics deviate significantly from a simple pseudo first-order model? (b) What is the kinetic mechanism of irreversible electrophilic co-substrate-mediated inactivation of human acidic GSTs? Inactivation of human placental GST π in the presence of 7-aminocephalosporanic acid, a non-electrophilic non-substrate ligand, is characterized and shown to occur via a process analogous to the second mechanism proposed for CDNB inactivation of the enzyme, namely: pH- and [ligand]-independent solvational inactivation.

Monoclonal Antibodies Against Human Placental Glutathione Transferase (Class п )

Five monoclonal antibodies (MAbs) were produced in a mouse hybridoma system against human placental glutathione transferase (GST pi). Four of these monoclonal antibodies, named 461 to 464, were of immunoglobulin G class, whereas the monoclonal antibody 465 was of IgA class. All these MAbs specifically recognized the glutathione transferase from human placenta (class pi) showing no cross reactivity against the basic and the neutral forms of GST from human liver. When each MAb was incubated with the GST pi, no inhibition of enzymatic activity towards 1-chloro-2,4-dinitrobenzene was observed except for MAb 465 which showed a slight inhibition to a serial dilution of 1:128.

Factors affecting the inactivation of human placental glutathione S-transferase π : The kinetic mechanism and pH-dependence of solvational and 1-chloro-2,4-dinitrobenzene-mediated inactivation of the enzyme

The kinetics of the inactivation of human placental GSH S-transferase π has been studied at 25° in the pH range 6.5 ⩽ pH ⩽ 9. At pH values ⩽7.0 the inactivation of GSH S-transferase π incubated in the absence of GSH and (i) in the absence or (ii) in the presence of CDNB (0−1.5 × 10 −3mol/dm 3) exhibited pseudo first-order kinetics with k obs for (i) and (ii) approximately equal (∼0.002 sec −1). The extent of inactivation in (i) approached a limiting value of 50% at infinite dilution of the enzyme; while in the presence of CDNB the extent of inactivation approached 100%. At any given pH such that 7 < pH ⩽ 9the pseudo first-order inactivation rate constant, k obs, exhibits a linear dependence on [CDNB] (Eqn 1): k obs = k 1 + k 2 [CDNB] where k 1 is invariant with pH and approximately equal to 0.002 sec −1. The first-( k 1) and second-( k 2)-order components of k obs suggest at least two mechanisms for the inactivation of GST by CDNB, these are: (i) a pH-invariant facilitation of solvational inactivation and (ii) a pH-dependent nucleophilic reaction of a thiol group (p k a = 8.85 ± 0.08) at or spatially close to the active site of the enzyme. A mechanistic rationale for the enzyme functioning as a dimer is discussed in detail.

Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site-directed fluorescent reagent

A fluorescent maleimide derivative, N-(4-anilino-1-naphthyl) maleimide (ANM), a specific probe for thiol groups, reacted with human placental glutathione transferase (GST, EC 2.5.1.18), causing a complete inactivation of the enzyme in a few minutes. The modified enzyme was denatured, alkylated and digested with (L-1-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin. The tryptic digest was analysed by HPLC and a fluorescent peptide was obtained. The sequence of this peptide allowed us, by a comparison with a well known primary structure, to assign the position 47 to the most reactive cysteine of GST enzyme.

Haem peptide–protein interactions. Part 3.—The kinetics and mechanism of the interaction between human placental glutathione S-transferase π and the non-substrate ligand microperoxidase-8 (MP-8)

The kinetics of the reaction between the haem-peptide microperoxidase-8 and human placental glutathione S-transferase π have been followed using the quenching of the MP-8 Soret absorbance, which is observed on mixing the two species. Under pseudo-first-order conditions ([MP-8]≫[GST]) and over a concentration range in which the MP-8 is monomeric in aqueous solution, the binding curves are biphasic. The rapid phase of binding is associated with a hydrophobic site at or close to the binding site for the co-substrate 1-chloro-2,4-dinitrobenzene (CDNB), while the slow-binding phase reflects hydrophobic interaction with or spatially close to the bilirubin binding site on the enzyme. The mechanism for the interaction has been shown to be of the random sequential form with perturbation due to solvational elimination of the fast site. These studies recommend MP-8 as a useful non-substrate ligand model for monomeric haemin with which to study the kinetics and mechanism of ligand binding to the glutathione S-transferases.

Studies on human placental glutathione S-transferase. Multiplicity and mother age influence

Human placental glutathione S-transferase was purified to apparent homogeneity by direct application of the crude homogenate into glutathione linked sepharose affinity chromatography. Chromatofocusing analysis in the presence of reduced glutathione resolved the enzyme into three acidic peaks eluted at pH 6.0, 5.7 and 5.5. About 36% of the initial activity was recovered in the isozyme fraction eluted at pH 6.0 whereas the isozymes eluted at pH 5.7 and 5.5 accounted for 20% and 25% of the activity respectively. Disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a single protein band in all the three separated isozymes. These isozymes were homodimers with an apparent relative molecular mass of 44.000 and subunit molecular mass of 21.000. The isozymes were immunologically related to each other and to the enzyme from goat and sheep placentae. Mother age had no influence in the placental glutathione S-transferase activity, albeit the activity was slightly higher in placenta obtained from younger women.

Haem peptide–protein interactions. Part 4.—The microperoxidase-8-mediated inhibition of the human placental glutathione S-transferase π catalysed conjugation of glutathione and 1-chloro-2,4-dinitrobenzene

The inhibition of the glutathione S-transferase π(GST) catalysed conjugation of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) by the haem octapeptide microperoxidase-8 (MP-8) has been investigated. Incubation of the enzyme with MP-8 results in a pseudo-first-order partial inactivation of the enzyme [kobs= 2.3 (±0.4)× 10–3 s–1; pH 6.5; 22.5 °C]. The kobs is identical to that found for dilutional inactivation of the enzyme observed in buffer alone. MP-8 increases only the extent of inactivation above that observed for dilutional inactivation.Incubation of the enzyme with varying concentrations of MP-8 was followed by a steady-state kinetic study of the enzyme-catalysed conjugation of GSH with CDNB at (a) fixed [GSH], varying [CDNB] and (b) fixed [CDNB] varying [GSH]. In case (a) the data conformed closely with mixed competitive/non-competitive inhibition kinetics (Ki= 3.2 × 10–7 mol dm–3, pH 6.5, 30 °C), while in case (b) the extent of inhibition was insufficient to allow analysis.These observations are rationalized and discussed in the light of the kinetic studies of MP-8 binding to GST π reported in the preceding paper.

Purification and biochemical characterization of the rabbit pulmonary glutathione S-transferase: Stereoselectivity and activity toward pyrene 4,5-oxide

Two homodimeric isozymes, glutathione S-transferase (GST) 25 kDa and GST 27 kDa, in equal proportion comprise the majority (>75%) of the pulmonary cytosolic GST of untreated rabbits. The subunits of GST 25 kDa and GST 27 kDa are distinguishable by electrophoretic mobility (25 and 27 kDa, respectively), apparent isoelectric points (p I 7.4 and p I 9.1, respectively), and immunoreactivity. Immunoblots indicated that these subunits may be minor components in hepatic cytosol. The pulmonary isozymes could not be distinguished by their activities toward chloro-2,4-dinitrobenzene (CDNB) or activity and stereoselectivity toward pyrene 4,5-oxide (PyO). The purified GST fractions represented ≤16% of the PyO activity for pulmonary cytosol. The stereoselectivity of the cytosolic GST for the pro- S-configured oxirane carbon of PyO was not maintained in the purified preparations which were virtually nonstereoselective. Immunoprecipitation of pulmonary cytosolic GST with anti-GST 27 kDa and anti-GST 25 kDa indicated that at least 84 and 60% of the activity toward CDNB and PyO, respectively, is mediated by the two isozymes. The specific PyO activities of GST 27 kDa, GST 25 kDa, and the rabbit hepatic preparations (~0.2 unit/mg) were similar to that of hepatic GST purified from horse, cow, and pig, and to human placental GST π (0.02 – 0.5 unit/mg) but one-tenth that of rat hepatic GST or human hepatic GST μ. However, the activity of the hepatic cytosol from rat and human was similar to that of rabbit. Thus, some GST isozymes may be particularly susceptible to modulation of activity/stereoselectivity that can be discerned with arene oxide substrates such as PyO.