Catalytic Activities of Human Alpha Class Glutathione Transferases

May 1, 2002 - The Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, SE−17177 Stockholm, Swede...
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Chem. Res. Toxicol. 2002, 15, 825-831

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Catalytic Activities of Human Alpha Class Glutathione Transferases toward Carcinogenic Dibenzo[a,l]pyrene Diol Epoxides† Kristian Dreij,‡ Kathrin Sundberg,‡ Ann-Sofie Johansson,§ Erik Nordling,|,⊥ Albrecht Seidel,# Bengt Persson,|,⊥ Bengt Mannervik,§ and Bengt Jernstro¨m*,‡ The Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, SE-17177 Stockholm, Sweden, Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-75123 Uppsala, Sweden, Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer Foundation, Lurup 4, DE-22927 Grosshansdorf, Germany, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden, and Stockholm Bioinformatics Center, Karolinska Institutet, SE-17177 Stockholm, Sweden Received February 12, 2002

In this study, human glutathione transferases (GSTs) of alpha class have been assayed with the ultimate carcinogenic (-)-anti- and (+)-syn-diol epoxides (DEs) derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-diol epoxide of the planar benzo[a]pyrene [(+)anti-BPDE] in the presence of glutathione (GSH). In all DEs, the benzylic oxirane carbon reacting with GSH, possess R-absolute configuration. GSTA1-1 demonstrated activity with all DEs tested whereas A2-2 and A3-3 only were active with the DBPDE enantiomers. With GSTA4-4, no detectable activity was observed. GSTA1-1 was found to be the most efficient enzyme and demonstrated a catalytic efficiency (kcat/Km) of 464 mM-1 s-1 with (+)-syn-DBPDE. This activity was about 7-fold higher than that observed with (-)-anti-DBPDE and more than 65-fold higher than previously observed with less complex fjord-region DEs. GSTA3-3 also demonstrated high kcat/Km with the DEs of DBP and a high preference for the (+)-syn-DBPDE enantiomer [190 vs 16.2 mM-1 s-1 for (-)-anti-DBPDE]. Lowest kcat/Km value of the active enzymes was observed with GSTA2-2. In this case, 30.4 mM-1 s-1 was estimated for (+)-synDBPDE and 3.4 mM-1 s-1 with (-)-anti-DBPDE. Comparing the activity of the alpha class GSTs with (-)-anti-DBPDE and (+)-anti-BPDE revealed that GSTA1-1 was considerable more active with the former substrate (about 25-fold). Molecular modeling studies showed that the H-site of GSTA1-1 is deeper and wider than that of GSTA4-4. This is mainly due to the changes of Ser212fTyr212 and Ala216fVal216, which cause a shallower active site, which cannot accommodate large substrates such as DBPDE. The higher activity of GSTA1-1 with (+)-synDBPDE relative to (-)-anti-DBPDE is explained by the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The presence of GSTA1-1 in significant amounts in human lung, a primary target tissue for PAH carcinogenesis, may be an important factor for the protection against the harmful action of this type of potent carcinogenic intermediates.

Introduction Polycyclic aromatic hydrocarbons (PAHs)1 are ubiquitous environmental contaminants known to induce mutations and tumors in experimental animals and, most † Part of this study was presented at the 18th International Symposium on Polycyclic Aromatic Compounds, The University of Cincinnati, Cincinnati, OH, September 9-13 2001. * To whom correspondence should be addressed. Phone: +46-8-7287576. Fax: +46-8-334467. E-mail: [email protected]. ‡ The Institute of Environmental Medicine. § Department of Biochemistry, Uppsala University. | Department of Medical Biochemistry and Biophysics, Karolinska Institutet. ⊥ Stockholm Bioinformatics Center, Karolinska Institutet. # Biochemical Institute for Environmental Carcinogens. 1 Abbrevations: PAHs, polycyclic aromatic hydrocarbons; GST, glutathione transferase; BP, benzo[a]pyrene; DBP, dibenzo[a,l]pyrene; (-)-anti-DBPDE, (11R,12S)-dihydroxy-(13S,14R)-epoxy-11,12,13,14tetrahydrodibenzo[a,l]pyrene; (+)-syn-DBPDE, (11S,12R)-dihydroxy(13S,14R)-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene; (+)-antiBPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; DE, diol epoxide; CDNB, 1-chloro-2,4-dinitrobenzene.

probably, also in humans (1-4). Most carcinogenic PAHs show as a structural feature a sterically hindered bayor fjord-region, the latter of which is associated with nonplanarity of the molecule (5-7) and in general with a more potent biological activity (8). Dibenzo[a,l]pyrene (DBP), an environmental contaminant (9) is the most carcinogenic PAH identified so far (Figure 1) (IUPAC nomenclature; dibenzo[def,p]chrysene or naphtho[1,2,3,4pqr]tetraphene) (10, 11). As with other PAHs, DBP requires metabolic activation to electrophilic intermediates and their subsequent covalent interaction with DNA to exert their biological effects. One route of activation common to all studied PAHs is the formation of diol epoxides (DEs) (2, 12, 13). In the case of DBP, the compound is stereoselectively bioactivated to (11S,12R)dihydroxy-(13S,14R)-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(+)-syn-DBPDE], and (11R,12S)-dihydroxy(13S,14R)-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(-)-anti-DBPDE] (14) by the sequential action of

10.1021/tx025519i CCC: $22.00 © 2002 American Chemical Society Published on Web 05/01/2002

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Figure 1. Structure and numbering system of the polycyclic aromatic hydrocarbons from which the diol epoxides used in this study are derived. The absolute stereochemistry of the syn- and anti-diol epoxides is shown to the right. The prefix syn indicates that the oxirane ring and the benzylic hydroxyl group are located on the same face of the molecule whereas anti indicates location of these groups on opposite faces.

CYP1A1/CYP1B1 and microsomal epoxide hydrolase (15). Unless the DEs are deactivated by cellular defense systems, the compounds may bind covalently to DNA, in particular to the exocyclic amino groups of the purine bases (3, 16). Studies employing mammalian cells have implicated a pivotal role for the soluble glutathione transferases (GSTs) in conjunction with glutathione (GSH) in protecting the genome against DE-induced damage and subsequent mutagenicity (17-20). Considering the high carcinogenic potency of DBP relative to other PAHs, such as benzo[a]pyrene (BP), and the potential risk the compound presents to human health, it seems essential to study to what extent human GSTs have the ability to detoxify DBP-derived DEs. Previous work with purified human GSTs and DEs derived from both bayand fjord-region PAHs (e.g., BP and benzo[c]chrysene, respectively) have shown that GSTA1-1, GSTM1-1, and two allelic variants of GSTP1-1 demonstrate significant activity (21-24). Furthermore, all enzymes seem to catalyze trans-addition of the glutathionylate anion to the benzylic oxirane carbon in the DE substrates. In contrast to GSTA1-1 and GSTM1-1, the GSTP1-1 variants exhibit an almost exclusive preference for the benzylic oxirane carbon with R-absolute configuration (22-25). Finally, human GSTA1-1 is more efficient than GSTM1-1 and GSTP1-1 in detoxifying the potent fjord region DEs of benzo[c]phenanthrene, benzo[c]chrysene and benzo[g]chrysene (21, 22). Recent work has shown that alpha class GSTs from mouse are highly active with fjord-region DEs (26, 27). In addition, mouse GSTA1-1 was found to be highly active with (7R,8S)-dihydroxy(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)anti-BPDE] (28). No information on the activity of human alpha class GSTs toward the more complex DEs of DBP is available and considering the potential biological importance in humans, we have in this study investigated the catalytic activities of GSTs A1-1, A2-2, A3-3, and A4-4 toward (+)-syn-DBPDE and (-)-anti-DBPDE. For comparative purposes, the activities of GSTs A1-1, A2-2, A3-3, and A4-4 toward (+)-anti-BPDE, the ultimate carcinogen of BP, have also been determined. Furthermore, molecular modeling has been performed in order to reveal factors governing substrate-enzyme interactions and catalytic efficiencies.

Materials and Methods Caution: Diol epoxides from polycyclic aromatic hydrocarbons are carcinogens, and thus experimental handling must be carried out under special safety conditions, e.g., those outlined in the NCI guidelines. Chemicals. Synthesis of the optically active syn- and antiDEs of BP and DBP was performed as previously described (21, 29, 30). Chromatographic standards of derivatives of BPDE and DBPDE were obtained as previously described (21, 24, 31). Glutathione Transferases. Recombinant human alpha class GSTs were obtained by expression in Escherichia coli and further purified as previously described (32-35). The specific activities of 80, 58, 23, and 6.5 µmol of CDNB/mg/min were used to calculate the amount of active protein of GSTs A1-1, A2-2, A3-3, and A4-4, respectively (33-36). Incubations. Enzyme corresponding to 5-200 µg of active protein/mL was incubated at 37 °C for 15 or 30 s with 2.5, 5, 10, 20, 40 µM DBPDE (added in dimethyl sulfoxide, final concentration 5%, v/v) and 5 mM GSH in 50 mM Tris-HCl buffer, pH 7.5 (final volume 100 µL), and analyzed for GSH conjugates by HPLC as described (24). For comparison, (+)-antiBPDE was incubated as described above but at 20 and 40 µM and catalytic efficiencies determined as before (21, 31). The rate of DBPDE solvolysis was determined as previously described for BPDE (21). Kinetic Data Analysis. Kinetic constants for DBPDE and GSTA were obtained from nonlinear regression analysis using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). Molecular Modeling. The atomic coordinates for GSTA1-1 and GSTA4-4 were retrieved from PDB entries 1GSE (37) and 1GUL (38), respectively. Hydrogens were added and idealized geometry was attained using the regularization procedure of the ICM software version 2.8 (39, 40) (Molsoft LLC, San Diego, CA). The side chain of Lys15 in 1GSE was changed back to Arg, as in the native protein, using the same rotamer for the side chain as for the replaced residue. Docking of substrates was performed with a flexible side-chain technique using Monte Carlo simulated annealing and conjugate-gradient minimization (41). The structures of the DBPDE diastereomers were obtained utilizing the molecular mechanical MM2 method (42) in Chem3D Ultra version 6.0 for Windows (CambridgeSoft Corporation, Cambridge, MA).

Results and Discussion GSTA-Catalyzed Conjugation of Diol Epoxides. Figure 1 shows the structure of the PAHs used in this study and the absolute configuration of the corresponding

Catalytic Activities of Alpha Class GSTs

Figure 2. Rate of GSH conjugation with (+)-syn-DBPDE (9) and (-)-anti-DBPDE (2) as a function of varying diol epoxide concentrations at a fixed saturated GSH concentration (5 mM) in the presence of GSTA1-1.

DEs. Each alpha class GST was incubated for short periods (15 or 30 s depending on the DE used) with varying concentrations of (-)-anti- or (+)-syn-DBPDE (2.5-40 µM) at a fixed saturating concentration of GSH (5 mM). The reaction was terminated by addition of alkaline mercaptoethanol and formation of conjugates estimated by HPLC as previously described (24). Example of the results obtained is shown in Figure 2. All reactions followed Michaelis-Menten kinetics and the kinetic constants calculated are summarized in Table 1. The most efficient alpha class enzyme is GSTA1-1 with a kcat/Km of 464 mM-1 s-1 toward (+)-syn-DBPDE. This activity is the highest ever observed for a human GST toward a DE substrate (cf. 43) and exceeds the values usually obtained with fjord-region DEs by 2 orders of magnitudes (21, 22). As evident from Table 1, the (-)anti-enantiomer is an inferior substrate as revealed by the 7-fold lower catalytic efficiency. The observed catalytic efficiencies of both GSTA1-1 and GSTA3-3 (see below) for catalyzing the conjugation of (+)-syn- and (-)anti-enantiomers of DBPDE with GSH has major implications for the overall detoxication of DBPDE due to the stereoselectivity involved in their formation from the parent, highly carcinogenic hydrocarbon DBP. As has been shown previously metabolic activation of DBP by CYP1A1 or CYP1B1 and microsomal epoxide hydrolase occurs via the (11R, 12R)-trans-dihydrodiol to result almost exclusively in the formation of (+)-syn- and (-)anti-DBPDE with R-configuration at the benzylic oxiranyl carbon (14, 15). Thus, there is a remarkable coordination between the stereoselectivity of detoxication of DBPDE enantiomers by GSTA1-1 and A3-3 and that of the metabolic activation of DBP to DBPDE. Previous results with other fjord-region DEs are consistent with a preference of GSTA1-1 for fjord region syn- and antiDEs with R-configured benzylic oxiranyl carbons, although not so pronounced as observed in the present study (21). Interestingly, the reverse enantioselectivity of GSTA1-1 in the detoxication of bay-region syn-DEs has been observed earlier (21). However, this has biologically much less importance, because among bay-region DEs such as BPDE, the carcinogenic activity is associated with the (+)-anti-enantiomer that shows again R-configuration at the benzylic oxiranyl carbon. In contrast, in the series of fjord-region DEs it appears that all four enantiomers show carcinogenic activity as has been demonstrated for benzo[c]phenanthrene (44). Compared to GSTA1-1, a more restrictive enantioselectivity has been found for GSTP1-1 (allelic variant Ile105) and the anti-DE enantiomers with R-configured benzylic carbon are in most cases the preferred substrates (22). In contrast, GSTM1-1 showed a much less pronounced enantioselectivity toward both bay- and fjord-region DEs

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investigated previously and the catalytic efficiencies were in the same order of magnitude as that observed for GSTA1-1 (22). This study shows that GSTA3-3 also demonstrated high activity toward DBPDE with a kcat/Km of 190 mM-1 s-1 toward (+)-syn-DBPDE and about 11-fold lower efficiency with the (-)-anti-enantiomer. Whereas no activity could be detected with GSTA4-4, GSTA2-2 expressed a moderate activity, 30.4 and 3.4 mM-1 s-1 with the (+)-syn- and (-)-anti-DBPDE, respectively. Data for the catalytic efficiencies with (+)-anti-BPDE are included for comparison. Relative to the activity observed with GSTA1-1 and (-)-anti-DBPDE, the stereochemical analogue and chemically more reactive (+)-anti-BPDE isomer is much poorer as substrate (see Table 1) (21). This is in great contrast to what has been observed with the corresponding mouse GSTA1-1 (>75% amino acid sequence and similar three-dimensional fold) (45). The human enzyme is at least 40-fold less active with (+)anti-BPDE (28, 46). It was suggested that in contrast to mouse GSTA1-1, the C-terminal helix is squeezed into the H-site in the human enzyme, thus rendering it more restricted and less accessible for (+)-anti-BPDE. In addition, it was suggested that Arg217 (position 216 in the mouse enzyme), which seems to play an important role in promoting the catalysis in mouse GSTA1-1, does not play a similar role in the human enzyme (45). The high efficiency of human GSTA1-1 with the large and bulky DBPDE, and in particular the (+)-syn-enantiomer, is not consistent with a restricted H-site. Furthermore, molecular modeling experiments (see below) suggest a promoting effect of Arg217 in the human GSTA1-1 mediated catalysis. Surprisingly, GSTA3-3 seems to be totally devoid of activity toward (+)-anti-BPDE despite its high amino acid identity (90%) with GSTA1-1 (34). The enzymes used in the present study are recombinant and assumed to fully reflect the properties of the native analogues. A recent study by Coles et al. (43) indicates however, that this might not be the case in all instances. It was shown that the activity of allelic variants of human GSTP1-1 differed between native and recombinant proteins, possibly due to differences in protein folding. If this also applies to GST alpha class enzymes is presently not known but seems to warrant investigation. Catalytic Efficiency and Diol Epoxide Structure. The results from this comparison reflect the structural differences between (+)-anti-BPDE and (-)-anti-DBPDE in addition to the amino acid composition and architecture of the H-sites in the enzymes. Both compounds have the same absolute configuration (R,S-diol, S,R-epoxide) and seem to preferentially adopt a conformation in which the hydroxyl groups are pseudo-diequatorially oriented (29, 47, 48). Moreover, the pyrenyl residue and the DE moiety is planar in BPDE whereas the benzo[e]pyrene aromatic system and the DE in DBPDE most likely is out of plane due to steric hindrance between H1 and H14, thus analogous to the situation with the phenanthryl residue in the DE of benzo[c]phenanthrene (49) (Figures 1 and 3).2 In fact, an angle of about 27° between the rings encompassing these positions in the parent compound has been determined (50). In addition to the difference in molecular geometry, DBPDE is expected to be consid2 Cho, K.-B., Dreij, K., Jernstro ¨ m, B., and Gra¨slund, A. Manuscript in preparation.

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Table 1. Kinetic Constants for Human GSTA Variants A1-1, A2-2, and A3-3 toward (-)-anti- and (+)-syn-DBPDEa Vmax (nmol/mg/min) GSTA1-1 GSTA2-2 GSTA3-3

Km (µM)

kcat/Km (mM-1s-1)

kcat (s-1)

(-)-anti-

(+)-syn-

(-)-anti-

(+)-syn-

(-)-anti-

(+)-syn-

(-)-anti-

(+)-syn-

653 ( 45 42.6 ( 3.4 429 ( 23

10541 ( 1504 675 ( 27 5813 ( 298

8.2 ( 1.5 10.5 ( 2.2 22.1 ( 2.4

18.9 ( 5.8 18.5 ( 1.5 25.5 ( 2.5

0.54 ( 0.04 0.036 ( 0.003 0.36 ( 0.02

8.8 ( 1.3 0.56 ( 0.02 4.8 ( 0.2

66.3 ( 12.7 (2.6)b 3.4 ( 0.8 (nd)c 16.2 ( 2.0 (nd)

464 ( 156 30.4 ( 2.7 190 ( 20

a Note: values are means ( standard error of at least three determinations. b Catalytic activity toward (+)-anti-BPDE with the same absolute configuration. c nd ) not detectable.

Table 3. Calculated Relative Activation/Binding Energies for the Substrate Pairs (+)-syn-DBPDE/(-)-anti-DBPDE and (-)-anti-DBPDE/(+)-anti-BPDE -∆∆Gb (kJ/mol)a enzyme

(+)-syn-DBPDE(A)/ (-)-anti-DBPDE(B)

(-)-anti-DBPDE(A)/ (+)-anti-BPDE(B)

GSTA1-1 GSTA2-2 GSTA3-3

5.0 5.7 6.3

8.3

a Calculated from the relationship ∆∆G ) -RT ln (k /K ) / b cat m A (kcat/Km)B.

Figure 3. Structures and preferred conformation of (+)-synDBPDE (top) and (-)-anti-DBPDE (bottom). Table 2. Rate of Solvolysis and Nonenzymatic Reaction of Diol Epoxides with GSH and the Calculated n-Octanol/ Water Distribution Coefficients (Kow) for the Aromatic Residue of the Diol Epoxides compd (-)-anti-DBPDE (+)-syn-DBPDE (+)-anti-BPDE

k2 t1/2 (min)a (mM-1 s-1) (×105)b ∆Edeloc/β log Kowe 25 16 1.5

34.7 58.0 28.0

0.713c 0.713c 0.794d

6.1 6.1 5.0

a Estimated from the rate of disappearance of diol epoxide at 37 °C in 50 mM Tris-HCl buffer, pH 7.5 (21). b Estimated from the rate of spontaneous GSH conjugate formation of diol epoxide at 37 °C in 50 mM Tris-HCl buffer, pH 7.5, and 5 mM GSH (21). c From ref 14. d From ref 51. e Calculated using ChemDraw Ultra 6.0.2 (C log P).

erably more lipophilic (see Table 2) due to the additional benzo ring and, due to the bent and helical structural distortion of the molecule, also more bulky. Previous work with human GSTA1-1 and less complex fjord-region DEs than DBPDE indicated that increased complexity of the aromatic ring system and thus, increased lipophilicity of the substrate, was associated with increased catalytic efficiency (21). Comparing the kcat/Km values calculated for R-configured anti-fjord region DEs (derived from benzo[c]phenanthrene, benzo[c]chrysene and benzo[g]chrysene) with those of the corresponding DBPDE reveals a difference of 20-100-fold in favor of the latter (21). Thus, extending the aromatic system in the DE from three rings (i.e., phenanthrene) to five (i.e., benzo[e]pyrene) greatly increases the catalytic efficiency of GSTA1-1. A correlation between increased lipophilicity and activity has been observed with mammalian GSTs and 4-hydroxyalkenals with an increasing number of

methylene groups (52). Accordingly, such factors, rather than the inherent chemical reactivity (k2 and ∆Edeloc/β) of the benzylic oxirane carbon (see Table 2), seem to promote the interaction and the rate of the subsequent conjugation reaction. This is further illustrated by calculating the incremental Gibbs free energy (-∆∆G) of transferring the benzo[e]pyrenyl vs the pyrenyl residue from a polar environment to the hydrophobic H-site of GSTA1-1. The value 8.3 kJ/mol (Table 3) is close to the value obtained from the ratio of the distribution coefficients (about 6.5 kJ/mol). As shown previously with GSTA1-1 and the fjordregion DEs discussed above, a marked preference for catalyzing S-glutathionylation of the (+)-syn-enantiomers was observed (21). This was also the case with DBPDE, and all active GSTA forms demonstrate an about 10-fold higher activity with the (+)-syn-enantiomer (this study). Thus, the change from R,S- to S,R-absolute configuration of the hydroxyl groups at the 11 and 12 positions greatly enhances the substrate turnover rate (Table 1). A closer look at the conformation of the molecule reveals however, that the hydroxy methine group at position 12 is localized at different sides of the aromatic moiety depending on whether the absolute configuration of the hydroxyl groups is R,S or S,R (Figure 3). In turn, this shift cause the oxiranyl function to change its spatial orientation and may in the case of the syn-DBPDE enantiomer expose the benzylic carbon more favorable for S-glutathionylation (see below). The relative change in -∆∆G was calculated to 5.0 kJ/mol (Table 3), which is close to the estimated π-value of hydrophobic-polar transfer of an aliphatic hydroxyl group (53). Similar values were calculated for GSTA2-2 and A3-3 (Table 3). Thus, conformational features of the DE residue as well as the spatial orientation of the hydroxyl groups are important determinants for the catalytic efficiency. Molecular Modeling of Diol Epoxides and GSTA. Molecular modeling clearly indicates that the main reason for the lack of activity with the DEs and GSTA4-4 is a more shallow and narrow H-site, which cannot accommodate substrates of this class. This is due to the fact that Tyr212 is pointing into the hydrophobic binding site and that the presence of Val216 creates a bulge that protrudes from the helix that covers the active site (38).

Catalytic Activities of Alpha Class GSTs

Figure 4. GSH conjugates of (+)-syn-DBPDE (A) or (-)-antiDBPDE (B) docked into the active site of GSTA1-1. The orientation in panel B is shifted 90° compared to panel A to allow for an easier viewing of the interactions. Arrows depict the hydrogen bond interactions (blue) and the unfavorable interactions (red) between DBPDE, GSH and protein. The lengths of the interactions are shown in angstroms. The carbon skeletons of the DBPDE-GSH conjugates are colored light-gray and the protein carbon atoms are colored dark-gray.

The docking calculations of free DBPDE enantiomers or their GSH conjugates in GSTA1-1 show that the large aromatic residue of DBPDE fits well into the H-site where it interacts with the aromatic side chains of Phe10 and Phe220. Ala12, Gly14, Leu41, Leu107, Met208, Ala216, Arg217, and Phe222 line the rest of the active site and create a hydrophobic cavity, large enough to accommodate the DBPDE molecules. Inspection of the GSH conjugate derived from (+)-syn-DBPDE reveals that all three hydroxyl groups are hydrogen-bonded to GSH or to the enzyme. The C11 hydroxyl group is hydrogenbonded to Arg217 Nη1 (distance 3.12 Å), the C12 hydroxyl to GSH γ-Glu O (distance 2.75 Å) and the C13 hydroxyl group is coordinated to Arg15 N (distance 2.54 Å) and Nη2 (distance 3.19 Å) (Figure 4A, blue arrows). In the case of the GSH conjugate derived from (-)-anti-DBPDE, only the C13 hydroxyl is weakly hydrogen-bonded to the GSH Gly N (distance 3.51 Å) (Figure 4B, blue arrow). The C12 of (-)-anti-DBPDE are located unfavorably close to the GSH Cys CR and GSH γ-Glu O (distance 3.03 and 2.18 Å, respectively). Furthermore, the C12 hydroxyl group makes an additional unfavorable contact with GSH γ-Glu O (distance 2.14 Å) (Figure 4B, red arrows). All these factors together may explain the lower activity observed with GSTA1-1 and (-)-anti-DBPDE. The 25-fold higher activity observed with (-)-antiDBPDE relative to (+)-anti-BPDE could in principle be

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explained by differences in hydrophobic interactions or repositioning of the substrate in the active site. The modeling experiments show that the BPDE-GSH conjugate exhibit similar interactions as the GSH conjugate of (-)-anti-DBPDE, thus only the C13 hydroxyl in DBPDE and the corresponding C9 hydroxyl in BPDE forms favorable interactions and with GSH Gly N (results not shown). The binding of (+)-anti-BPDE in human GSTA1-1 involves apparently a different set of interactions relative to those following binding in mouse GSTA1-1 (45). In this case the interactions are more close to those observed with (+)-syn-DBPDE in this study. Evidently, a more conclusive mechanistic description requires structural studies by X-ray diffraction analysis. Significance of GSTA-Catalyzed Diol Epoxide Detoxication in Vivo. Due to the ubiquitous formation and distribution of PAH in the environment, the human exposure is extensive. At this stage we can only speculate on the role of alpha class GSTs catalyzed detoxication of potentially carcinogenic DBPDE intermediates in humans. The sites of entry of PAHs to the human body are the respiratory and digestive tracts and the skin. A dominant contribution to the overall exposure of PAH is via the food chain, and this is also the case for cigarette smokers (54, 55). Although smokers in many cases exhibit larger levels of adducts relative to the level observed in nonsmokers, the difference is far from what to anticipate (56-58). This raises several questions with regard to the sites of bioactivation of the parent PAH to the ultimate DNA-binding intermediates (i.e., DEs), the transport from the site of formation to DNA in distal tissues and the distribution and level of expression of enzymes involved in detoxication (i.e., GSTs). The major site of xenobiotic metabolism is the liver, and independent of the site of PAH uptake, most of the compounds will be metabolized in this organ. Accordingly, PAH derived DNA adducts found in extra-hepatic tissues, such as the lung, result in part from leakage of proximal and/ or ultimate DNA-binding intermediates from the liver into the systemic circulation and subsequent transport and DNA adduct formation at distant sites as has been demonstrated in the rat (59). With respect to protection against DNA-binding intermediates, this scenario requires a focus on detoxication systems not only in target tissues but also at the primary site of bioactivation. Great interest and much work have been devoted to studies on the role of GSTM1-1 in protection against PAH-induced carcinogenesis in humans. About 50% of the Caucasian population lack the GSTM1 gene and may be at a higher risk relative to those expressing the enzyme. The results obtained so far, although not conclusive, indicate that individuals expressing GSTM1-1 may be less sensitive to develop lung cancer following PAH exposure than nonexpressing individuals. Since GSTM1-1 is present in substantial amounts in the liver but not, or at very low levels, in the lung, these findings are compatible with a role of the liver in lung carcinogenesis. GSTA1-1 is the dominating GST in the liver and the enzyme is also present in the lung (60, 61). GSTA2-2 is also present in these tissues but at much lower levels whereas GSTA3-3 and GSTA4-4 is present primarily in placenta (62) and brain (33, 62), respectively. Considering the extraordinary high activity of human alpha class GSTs, and in particular the A1-1 isoenzyme, toward the extremely potent carcinogenic DBPDE enantiomers, both the presence and the level of expression may be an

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important determinant for an individuals susceptibility to this type of compounds.

Acknowledgment. This study was supported by grants from Swedish Match, the Swedish Research Council, Swedish Cancer Society, and the Swedish Foundation for Strategic Research. The authors acknowledge the skillful work of Dr. Andreas Luch synthesizing the optically active DBPDE.

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