Role of arginine 216 in catalytic activity of murine Alpha class glutathione transferases mGSTAl-1 and mGSTA2-2 toward carcinogenic diol epoxides of polycyclic aromatic hydrocarbons
Ajai Pal,
Yijun Gu,
Christian Herzog1,,
Sanjay K. Srivastava,
Piotr Zimniak1,2,,
Xinhua Ji3, and
Shivendra V. Singh4,
Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261,
1 Departments of Medicine and Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences and
2 McClellan VA Hospital Medical Research, Little Rock, AR 72205 and
3 Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA
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Abstract
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Murine class Alpha glutathione (GSH) transferase A1-1 (mGSTA1-1) is unique among mammalian Alpha class GSTs due to its exceptionally high catalytic activity toward (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated metabolite of an environmentally relevant carcinogen, benzo[a] pyrene (BP). However, the molecular basis for high catalytic activity of mGSTA1-1 toward (+)-anti-BPDE is not clear. In the present study, we demonstrate that an arginine residue at position 216, which is conserved in some but not all mammalian class Alpha GSTs, plays an important role in catalytic activity of mGSTA1-1 toward (+)-anti-BPDE and carcinogenic diol epoxides of other environmentally relevant polycyclic aromatic hydrocarbons (PAHs). The catalytic efficiency (kcat/Km) of mGSTA1-1 for the GSH conjugation of (+)-anti-BPDE (108/mM/s) was reduced by about 58% upon replacement of arginine 216 with alanine (R216A). This was mainly due to a significantly lower Vmax for the R216A mutant of mGSTA1-1 compared with wild-type mGSTA1-1. The R216A mutation also resulted in a statistically significant reduction (>70%) in specific activity of mGSTA1-1 toward racemic anti-diol epoxides of chrysene and benzo[c]phenanthrene (anti-CDE and anti-B[c]PDE, respectively). The catalytic activity of mGSTA2-2, which is a close structural homologue of mGSTA1-1, was also reduced upon R216A mutation. The results of the present study clearly indicate that an arginine residue at position 216 is critical for catalytic activity of mGSTA1-1 and mGSTA2-2 toward carcinogenic diol epoxide metabolites of various PAHs that are abundant in the environment and suspected human carcinogens.
Abbreviations: (+)-anti-BPDE, (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene; anti-B[c]PDE, anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; anti-CDE, anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene; BP, benzo[a]pyrene; GSBpd, GSH conjugate of (+)-anti-BPDE; GSH, glutathione; GST, glutathione transferase; mGSTA1-1, murine class Alpha GST isoenzyme A1-1; mGSTA1-1/R216A; arginine 216
alanine mutant of mGSTA1-1; mGSTA1-1GSH, mGSTA1-1 in complex with GSH; mGSTA1-1GSBpd, mGSTA1-1 in complex with GSBpd; mGSTA2-2, murine class Alpha GST isoenzyme A2-2; mGSTA2-2/R216A, arginine 216
alanine mutant of mGSTA2-2; hGSTA1-1, human class Alpha GST isoenzyme A1-1; H-site, hydrophobic substrate binding site; PAHs, polycyclic aromatic hydrocarbons; R216A, enzyme with mutation of arginine 216 with alanine.
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Introduction
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Benzo[a]pyrene (BP) is the prototypical and best studied member of the polycyclic aromatic hydrocarbon (PAH) family of environmental pollutants that are thought to be etiological factors for chemically induced cancers in humans (1). Tumorigenic activity of BP is mainly due to its diol epoxide isomer (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] (2,3), which is generated through mediation of cytochrome P450-dependent mono-oxygenase and epoxide hydrolase (4,5). While covalent interaction of (+)-anti-BPDE with nucleophilic sites in DNA is believed to be critical for BP-induced tumorigenesis (5,6), several different mechanisms exist that can inactivate (+)-anti-BPDE and consequently prevent its interaction with DNA (716). The known mechanisms of (+)-anti-BPDE inactivation include spontaneous hydrolysis, hydration by epoxide hydrolase and glutathione (GSH) transferase (GST)-catalyzed conjugation with GSH (712). In addition, riboflavin 5'-phosphate, several naturally occurring plant phenols such as ellagic acid and certain phenolic metabolites of BP have been shown to inhibit mutagenic and/or tumorigenic activity of anti-BPDE (1316). Mechanistic studies have revealed that while riboflavin 5'-phosphate increases the rate of conversion of anti-BPDE to tetrols without forming covalent adducts (13), ellagic acid forms covalent adducts with this diol epoxide (15). However, the GST-catalyzed conjugation of (+)-anti-BPDE with GSH is believed to be the most important enzymatic pathway for its cellular detoxification (1012). Recent studies, including those from our laboratory, have shown that overexpression of GSTs in cells, through stable transfection, reduces the formation of (+)-anti-BPDEDNA adducts (17,18).
Glutathione transferases are a superfamily of multifunctional isoenzymes that can detoxify a wide variety of electrophilic xenobiotics primarily by catalyzing their conjugation with GSH (19,20). Cytosolic GST activity in mammalian tissues is due to multiple isoenzymes arising from dimeric combinations of either identical (homodimer) or structurally different subunits (heterodimer). Cytosolic GSTs have been grouped into several classes, namely Alpha, Mu, Pi (21), Theta (22), Sigma (23), Kappa (24) and Zeta (25), based on their structural and catalytic characteristics. GST isoenzymes of the above classes exhibit overlapping yet distinct substrate specificity (1921).
Recent studies from our laboratory have shown that murine class Alpha isoenzyme mGSTA1-1, unlike rat and human class Alpha GSTs (10,11), is exceptionally effective in catalyzing the GSH conjugation of (+)-anti-BPDE (26). The catalytic efficiency (kcat/Km) of mGSTA1-1 toward (+)-anti-BPDE is approximately 3.3-, 77- and 655-fold higher compared with murine class Alpha isoenzymes mGSTA2-2, mGSTA3-3 and mGSTA4-4, respectively (26). However, the molecular basis for high catalytic activity of mGSTA1-1 toward (+)-anti-BPDE is not fully understood.
Recently, we have solved the crystal structures of mGSTA1-1 in complex with GSH (mGSTA1-1GSH) as well as in complex with the GSH conjugate of (+)-anti-BPDE (mGSTA1-1GSBpd) (27). The structural comparison between mGSTA1-1GSH and mGSTA1-1GSBpd reveals dramatic positional displacement and conformational change of an arginine residue in position 216 upon substrate binding. We found that the side chain of arginine 216 (R216) points away from the hydrophobic substrate-binding site (H-site) in mGSTA1-1GSH complex but probes into the active site in the mGSTA1-1GSBpd structure (27). These observations led us to hypothesize that R216 may be critical for high catalytic activity of mGSTA1-1 toward (+)-anti-BPDE. In the present study, we have experimentally verified this hypothesis by determining the effect of mutation of arginine 216 with alanine (R216A) on catalytic efficiency of mGSTA1-1 toward (+)-anti-BPDE. The results of the present study reveal that replacement of R216 with alanine dramatically reduces the activity of mGSTA1-1 toward (+)-anti-BPDE as well as carcinogenic diol epoxides of other PAHs. A similar mutation also reduces catalytic activity of mGSTA2-2, which shares ~95% amino acid sequence identity with mGSTA1-1. Our results clearly indicate that an arginine residue in position 216 plays an important role in catalytic activity of mGSTA1-1 and mGSTA2-2 toward PAH-diol epoxides.
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Materials and methods
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Molecular modeling
Molecular modeling was carried out using program suites X-PLOR (28) and O (29) on an SGI Indigo2 Impact 10000 workstation. Initial model of the R216A mutant of mGSTA1-1 (mGSTA1-1/R216A) in complex with GSBpd (mGSTA1-1/R216AGSBpd) was built based on the crystal structure of wild-type mGSTA1-1 in complex with GSBpd (mGSTA1-1GSBpd) (27). A homodimer of mGSTA1-1/R216AGSBpd was constructed and subjected to geometry optimization using the conjugate gradient method of Powell (30). All solvent molecules from the crystal structure were excluded in energy minimization. The Engh and Huber (31) geometric parameters were used as the basis of the force field.
Site-directed mutagenesis
mGSTA1 and mGSTA2 cDNA clones in the bacterial expression vector pET-11d (26) were subjected to site-directed mutagenesis using the Quick Change kit (Stratagene, La Jolla, CA). To introduce the R216A mutation, the oligonucleotides 5'-ATT CAA GAA GCA GCC AAG GCT TTC AAG and 5'-ATT GAA GAA GCA GCC AAG GTT TTC AAG (and their corresponding reverse-complemented oligonucleotides) were used for mGSTA1-1 and mGSTA2-2, respectively. The mutated codons are underlined. Mutations were confirmed by sequencing and the resulting plasmids were used to transform Escherichia coli BL21(DE3)pLysS (Novagen, Madison, WI) for protein expression.
Bacterial culture and GST purification
Cultures of E.coli BL21(DE3)pLysS transformed with pET-11d carrying either wild-type or mutated mGSTA1 or mGSTA2 coding sequences were grown overnight in LuriaBertani (LB) media (Life Technologies, Gaithersburg, MD) containing 100 µg/ml ampicillin (Sigma, St Louis, MO). Overnight cultures were diluted 1:50 with LB media and incubated further with shaking for 2 h at 37°C. IPTG (Sigma) was then added to a final concentration of 2 mM, followed by incubation with shaking for an additional 34 h. Subsequently, bacteria was harvested by centrifugation at 800 g for 5 min and kept frozen at 80°C until use. In general, 400500 ml bacterial culture was used for the purification of GSTs. The bacterial pellet was thawed, re-suspended in 50 mM TrisHCl pH 8.0 containing 5 mM EDTA and 50 µg/ml lysozyme and incubated at room temperature for 15 min. The sample was sonicated to shear genomic DNA. Bacterial lysate was centrifuged at 14 000 g for 30 min. The supernatant fraction was dialyzed overnight against affinity buffer (22 mM potassium phosphate buffer, pH 7.0, containing 1.4 mM 2-mercaptoethanol) and subjected to affinity chromatography on GSH linked to epoxy-activated Sepharose 6B (Sigma). GSH affinity chromatography was performed by the method of Simons and Van der Jagt (32) with some modifications previously described (33). GST was eluted with 5 mM GSH in 50 mM TrisHCl pH 9.5 containing 1.4 mM 2-mercaptoethanol. Purification of GST was monitored by determining enzyme activity toward 1-chloro-2,4-dinitrobenzene as described by Habig et al. (34). Protein content was measured by the Bradford method (35).
GST activity determination
GST activity toward (+)-anti-BPDE was determined as described by us previously (12,26). Briefly, the reaction mixture in a final volume of 0.1 ml contained 50 mM TrisHCl pH 7.5 containing 2.5 mM KCl and 0.5 mM EDTA (TKE buffer), 2 mM GSH, 15 µg/ml GST protein and desired concentration of (+)-anti-BPDE (Chemsyn Science, Lenexa, KS). The reaction was initiated by adding (+)-anti-BPDE and the reaction mixture was incubated at 37°C for 30 s. The reaction was stopped by rapidly mixing with 0.1 ml cold acetone, followed by extraction twice with ethyl acetate saturated with TKE buffer. The GSH conjugate of (+)-anti-BPDE in the aqueous phase was quantified by reverse-phase HPLC as previously described (12,26). A blank without the GST protein was included to account for non-enzymatic GSH conjugation of (+)-anti-BPDE. GST activity was determined as a function of varying (+)-anti-BPDE concentration (10120 µM) at a fixed saturating concentration of GSH (2 mM) to determine the kinetic constants. Kinetic constants were determined by non-linear regression analysis of experimental data points using the MichaelisMenten equation. Specific activities of wild-type mGSTA1-1 and mGSTA2-2, and their respective R216A mutants toward racemic anti-CDE and anti-B[c]PDE (Midwest Research Institute, Kansas City, MO) were determined using 2 mM GSH as previously described (3638). Concentrations of anti-CDE and anti-B[c]PDE in the reaction mixture were 120 and 320 µM, respectively.
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Results and discussion
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Figure 1
shows the results of molecular modeling of active site of R216A mutant of mGSTA1-1 (mGSTA1-1/R216A) in complex with GSBpd (mGSTA1-1/R216AGSBpd), which was built based on the crystal structure of the mGSTA1-1GSBpd complex (27). Comparison of the model with the crystal structure reveals that the conjugated ring system of GSBpd in mGSTA1-1/R216AGSBpd is roughly located at a similar position as in the mGSTA1-1GSBpd complex structure (Figure 1
). In the crystal structure of mGSTA1-1GSBpd, the BPD moiety of the product molecule is sandwiched between the side chain of R216 and the phenyl ring system of F9 (27), whereas the BPD moiety is no longer parallel to the phenyl ring system of F9 in the model of mGSTA1-1/R216AGSBpd (Figure 1
). Instead it tilts about 20° with respect to the F9 side chain (Figure 1
). Thus, the binding of the substrate/intermediate may be weakened and the proper position/ orientation of the substrate/intermediate may be misarranged, both of which are expected to lower catalytic activity of mGSTA1-1/R216A toward (+)-anti-BPDE. In addition to the contribution in substrate binding, R216 is believed to assist in the ring opening of the epoxide (27). Therefore, R216A mutation is also likely to reduce the activity of mGSTA1-1 toward (+)-anti-BPDE due to elimination of the electrostatic assistance of R216 in the ring opening reaction of the epoxide.

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Fig. 1. Stereo view of the hydrophobic substrate binding site (H-site) of mGSTA1-1GSBpd (yellow-orange; ref. 27) superimposed with the model of mGSTA1-1/R216AGSBpd (blue, present study). The illustration was prepared with RIBBONS (43).
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To test the above possibilities, we have determined the effect of the R216A mutation on catalytic activity of mGSTA1-1 toward (+)-anti-BPDE and the results are shown in Figures 2 and 3
and Table I
. Figure 2
depicts reverse-phase HPLC analysis of water soluble products resulting from the reaction of 2 mM GSH and 120 µM (+)-anti-BPDE in the presence of 15 µg/ml wild-type mGSTA1-1 and mGSTA2-2, and their respective R216A mutants. As can be seen in Figure 2
, the GSH conjugation of (+)-anti-BPDE was markedly higher in the presence of wild-type mGSTA1-1 compared with wild-type mGSTA2-2, which is in agreement with our earlier observations (26). Furthermore, R216A mutation resulted in a marked decrease in the formation of GSH-(+)anti-BPDE conjugate for both mGSTA1-1 and mGSTA2-2 (Figure 2
).

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Fig. 2. Reverse-phase HPLC analysis of water soluble products resulting from the reaction of 2 mM GSH and 120 µM (+)-anti-BPDE in the presence of 15 µg/ml wild-type mGSTA1-1 and mGSTA2-2 and their respective R216A mutants.
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Table I. Kinetic constants for wild-type mGSTA1-1 and mGSTA2-2 and their respective R216A mutants toward (+)-anti-BPDE
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As shown in Figure 3
, both wild-type mGSTA1-1 and its R216A mutant adhered to MichaelisMenten kinetics when GST activity was measured as a function of varying (+)-anti-BPDE concentration (10120 µM) at a fixed saturating concentration of 2 mM GSH. Kinetic constants for wild-type mGSTA1-1 and its R216A mutant are summarized in Table I
. Mutation of R216 with alanine resulted in a statistically significant decrease (~67%; P < 0.05) in the Vmax of the enzyme. The Km values for the wild-type mGSTA1-1 and mGSTA1-1/R216A mutants were not statistically significantly different. The catalytic efficiency (kcat/Km) of wild-type mGSTA1-1 (108/mM/s) was reduced markedly (~58%) upon mutation of R216 with alanine (Table I
). These results clearly indicate that R216 facilitates mGSTA1-1 catalyzed GSH conjugation of (+)-anti-BPDE.
mGSTA2-2 is a close homologue of mGSTA1-1 and contains an arginine at position 216 (26). However, mGSTA1-1 is >3-fold more efficient than mGSTA2-2 in catalyzing the GSH conjugation of (+)-anti-BPDE (26). To determine if arginine in position 216 plays a role in the activity of mGSTA2-2 toward (+)-anti-BPDE, the influence of R216A mutation on catalytic activity of this isoenzyme was also investigated. As shown in Table I
, the kcat/Km of mGSTA2-2 toward (+)-anti-BPDE was reduced only by 45% upon replacement of arginine 216 with alanine. Similar to mGSTA1-1, a reduction in the Vmax value accounted for the lower kcat/Km of the mutant enzyme compared with wild-type mGSTA2-2 (Table I
).
The effects of R216A substitution on catalytic activities of mGSTA1-1 and mGSTA2-2 toward anti-diol epoxides of chrysene and benzo[c]phenanthrene (anti-CDE and anti-B[c]PDE, respectively), which are other members belonging to the PAH family of environmental carcinogens (5), were also investigated and the results are shown in Table II
. The specific activities of mGSTA1-1 toward anti-CDE and anti-B[c]PDE were reduced by about 72 and 74%, respectively, due to the R216A mutation (Table II
). The specific activities of mGSTA2-2/R216A toward anti-CDE and anti-B[c]PDE were lower by about 65 and 32%, respectively, compared with wild-type mGSTA2-2 (Table II
).
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Table II. Specific activities of wild-type mGSTA1-1 and mGSTA2-2 and their respective R216A mutants toward racemic anti-diol epoxides of chrysene and benzo[c]phenanthrene
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The results of the present study reveal that the replacement of R216 with alanine causes a dramatic reduction in catalytic efficiency of mGSTA1-1 toward (+)-anti-BPDE. Structural studies from our laboratory have shown that the H-site of mGSTA1-1 is composed of various conserved N-terminal residues including Y8, F9 and R14, and the C-terminal residues M207, A215, F219 and I221 (27). The side chains of these residues create a hydrophobic pocket for the binding of xenobiotic substrates, such as (+)-anti-BPDE. The contacts between the BPDE moiety and the H-site residues include both electrostatic and hydrophobic interactions (27). The active site of mGSTA1-1 seems to undergo significant conformational changes upon the binding of GSBpd. Noticeably, the C
atom of R216 moves ~2.3 Å and the side chain swings from pointing away from the H-site to protruding into the active center (27). Moreover, the guanidinium group of R216 travels ~7.7 Å and forms a strong hydrogen bond (2.76 Å) with the C8 hydroxyl group of BPDE. This electrostatic interaction may serve to orient and position the substrate (+)-anti-BPDE in the H-site (27). In addition, this interaction may also facilitate the ring opening reaction of the epoxide, which is the rate-limiting step in GST-catalyzed GSH conjugation of epoxide substrate (20). As shown in Figure 1
, the R216A mutation may disrupt these interactions and reduce the catalytic activity of the protein toward (+)-anti-BPDE. The results of the present study are consistent with the predictions from molecular modeling studies.
Environmentally relevant PAH-diol epoxides are categorized into two classes, bay-region and fjord-region, depending upon the location of the epoxide function. The bay-region class PAH-diol epoxides (e.g. anti-CDE and anti-BPDE) are planar, whereas fjord-region diol epoxides, such as anti-B[c]PDE, are distorted from planarity due to the location of their epoxide functional group in a sterically crowded region (5). The fjord-region class PAH-diol epoxides are more potent carcinogens in animal tumor bioassays relative to bay-region type diol epoxides (5). The results of the present study reveal that R216A mutation also causes a statistically significant reduction in the specific activity of mGSTA1-1 toward anti-CDE as well as anti-B[c]PDE. Our results suggest that R216 may be important in mGSTA1-1-catalyzed GSH conjugation of not only bay-region PAH diol epoxides, but also the PAH-diol epoxides belonging to the fjord-region class.
The present study demonstrates that the activity of mGSTA2-2, which is structurally very similar to mGSTA1-1 (26), toward (+)-anti-BPDE is also reduced upon R216A mutation. It is important to point out that mGSTA1-1 is >3-fold more efficient than mGSTA2-2 in catalyzing the GSH conjugation of (+)-anti-BPDE (26). The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions, including five residues in the C-terminal region (amino acid residues in positions 207, 213, 218, 221 and 222) (26). Previous studies from our laboratory (39) have shown that methionine 207 and isoleucine 221 are critical for relatively higher activity of mGSTA1-1 toward (+)-anti-BPDE compared with that of mGSTA2-2 (corresponding residues in mGSTA2-2 are leucine and phenylalanine, respectively). The results of the present study reveal that R216 plays an important role in catalytic activity of mGSTA2-2 toward PAH diol epoxides.
Another interesting aspect of the present study is that the specific activity of wild-type mGSTA2-2 toward racemic anti-B[c]PDE is ~2.5-fold higher compared with wild-type mGSTA1-1. This is particularly surprising since mGSTA1-1 is relatively more active than mGSTA2-2 toward every PAH-diol epoxide substrate tested to date, including some fjord-region class diol epoxides (26,40). For example, we have shown previously that mGSTA1-1 is ~2.3-fold more efficient than mGSTA2-2 in the GSH conjugation of fjord-region ()anti-diol epoxide isomer of benzo[g]chrysene (40). As shown in Table II
, the specific activity of mGSTA1-1 toward anti-CDE, which is a bay-region class PAH diol epoxide, is more than 4-fold higher than that of mGSTA2-2. Collectively, the results of our studies suggest that while mGSTA1-1 plays an important role in the inactivation of bay-region PAH-diol epoxides, mGSTA2-2 may be important in GSH conjugation of some fjord-region class PAH diol epoxides, such as anti-B[c]PDE.
Human GSTA1-1 (hGSTA1-1) has a very low activity toward (+)-anti-BPDE compared with mGSTA1-1 (11,26). This is particularly surprising considering that hGSTA1-1 contains an arginine at position 216 (41) and that mGSTA1-1 and hGSTA1-1 have similar three-dimensional folds (27,42). The H-site in hGSTA1-1 can accommodate various bay- and fjord-region diol epoxides and catalyze their GSH conjugation at an appreciable rate (11). However, when compared with the H-site in mGSTA1-1, the C-terminal helix appears to squeeze into the H-site in hGSTA1-1 and is probably kept in that position by a salt bridge between R220 and D41 (27). Consequently, relative to mGSTA1-1, the H-site of hGSTA1-1 is too narrow to accommodate bulky substrates such as (+)-anti-BPDE. The unavailability of R216 and narrow H-site are probably responsible for the very low catalytic efficiency of hGSTA1-1 toward (+)-anti-BPDE as compared with mGSTA1-1.
In conclusion, the results of the present study clearly indicate that an arginine residue at position 216 is important in the catalytic activity of mGSTA1-1 and mGSTA2-2 toward (+)-anti-BPDE as well as activated metabolites of other environmentally relevant PAHs.
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Notes
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4 To whom correspondence should be addressedEmail: singhs{at}msx.upmc.edu 
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Acknowledgments
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This investigation was supported in part by USPHS grants CA76348 and CA55589 (to S.V.S.) awarded by the National Cancer Institute.
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Received February 12, 2001;
revised April 17, 2001;
accepted April 27, 2001.