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Analysis of the Kinetic Mechanism of Haloalkane. Conjugation by Mammalian θ-Class Glutathione. Transferases. F. Peter Guengerich,* W. Andrew McCormic...
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Chem. Res. Toxicol. 2003, 16, 1493-1499

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Analysis of the Kinetic Mechanism of Haloalkane Conjugation by Mammalian θ-Class Glutathione Transferases F. Peter Guengerich,* W. Andrew McCormick, and James B. Wheeler† Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232-0146 Received July 21, 2003

Glutathione (GSH) transferases (GSTs) catalyze the conjugation of small haloalkanes with GSH. In the case of dihalomethanes and vic-1,2-dihaloalkanes, the reaction leads to the formation of genotoxic GSH conjugates. A generally established feature of the reaction of the mammalian θ-class GSTs, which preferentially catalyze these reactions, is the lack of saturability of the rate with regard to the substrate concentration. However, the bacterial GST DM11 catalyzes the same reactions with a relatively low Km. Recently, DM11 has been shown to exhibit burst kinetics, with a rate-determining koff rate for product (Stourman et al. (2003) Biochemistry 42, 11048-11056). We examined rat GST 5-5 and human GST T1-1 and did not detect any burst kinetics in the conjugation of C2H5Cl, CH2Br2, or CH2Cl2, distinguishing these enzymes from GST DM11. The kinetic results were fit to a minimal mechanism in which the rate-limiting step is halide displacement. The differences in the steady state kinetics of conjugations catalyzed by bacterial GST DM11 and the mammalian GSTs 5-5 and T1-1 are concluded to be the result of differences in the rate-limiting steps and not to inherent enzyme affinity for the haloalkanes. The results may be interpreted in the context of a model in which the halide order affects the rate of carbon-halogen bond cleavage of all such reactions catalyzed by the GSTs. With GST DM11, the halide order is manifested in the Km parameter but not kcat. With mammalian GSTs, the high Km is difficult to estimate. With all of the GSTs, the halide order is seen in the enzyme efficiency, kcat/Km, with C-Br cleavage ∼10-fold faster than C-Cl cleavage. The ratio kcat/Km is the most relevant parameter for issues of risk assessment.

Introduction Haloalkanes can produce a variety of toxicities at high doses. One compound of interest is CH2Cl2, a high volume commodity chemical (1) that can cause liver and lung tumors in mice when administered at high doses by gavage (2, 3). An issue is the relevance of the mouse results, in consideration of the lack of cancer in rats treated with the same doses (4-6). The genotoxic activity of CH2Cl2 is related to bioactivation by GST.1 Conjugation of CH2Cl2 or other methylene dihalides proceeds as shown in Scheme 1 (7). The GSCH2X intermediate is highly unstable (8, 9), and the only direct evidence for its formation comes from 19F NMR studies with CH2ClF, yielding the somewhat more stable GSCH2F (10). An intermediate could not be detected in the reaction of 13CH2Cl2 (11). Recently, we have demonstrated the formation of deoxyribonucleoside adducts to which the elements of GSCH2 have been added following reaction of CH2Cl2 and CH2Br2 with GSTs.2 The risk assessment of CH2Cl2 involves the issue of dose dependence. In early work on the pharmacokinetics * To whom correspondence should be addressed. Tel: (615)322-2261. Fax: (615)322-3141. E-mail: [email protected]. † Present address: Alcon Laboratories, 6201 S. Freeway, R3-22, Fort Worth, TX 76134. 1 Abbreviation: GST, GSH transferase. 2 Marsch, G. A., Botta, S., Martin, M. V., McCormick, W. A., and Guengerich, F. P. Submitted for publication.

and carcinogenicity of CH2Cl2, the observation was made that tumor formation was not readily saturable with increasing doses of CH2Cl2 (4, 12). This information was associated with the lack of saturability of the GSTcatalyzed conversion of CH2Cl2 to HCHO to suggest a role for the GST reaction in genotoxicity (13), which was supported by bacterial mutagenicity studies (14-21). The conjugation of CH2Cl2 was shown to be attributable primarily to the θ-class GSTs 5-5 in rat and T1-1 in human (18, 21-23). A mouse θ-class enzyme also catalyzes the reaction (24). In contrast to the lack of saturability of the GST reaction, liver microsomal cytochrome P450 (primarily 2E1) (25) oxidizes CH2Cl2 to CO (26) with a low Km (12, 13, 27). The apparent low affinity of the mammalian GSTs for CH2Br2 and other methylene dihalides (e.g., Km of 110 mM for CH2Cl2 for GST 5-5 (21)) contrasts with the bacterial GST DM11 (Km of 45 µM (21)), which has a low Km for CH2Cl2 and provides Methylophilus species the ability to grow on CH2Cl2 as a sole carbon source (21, 28). The kinetic behavior of mammalian GSTs is also an issue in that evaluation of the relative toxicities of individual methylene dihalides (e.g., in physiologically based pharmacokinetic models (4, 12, 13, 29)) requires an appropriate understanding of what parameters are most appropriate. In previous work (21, 30), we demonstrated that the lack of saturability of the conversion of methylene diha-

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Scheme 1. Conjugation of CH2X2 (X ) Halogen) with GSH Catalyzed by GSTa

a

The intermediate GS+ ) CH2 is a possibility, but at this time, no experimental evidence has been presented.

lides to HCHO was not a phenomenon peculiar to that reaction, which is unusual in that the substrate GSH is regenerated (Scheme 1), but was also seen in the conjugations of 1,2-vic-dihaloalkanes and monohaloethanes. Subsequent work by Stourman et al. (9) with GST DM11 has demonstrated burst kinetics in the conjugation of C2H5Br. This result indicates that a step following product formation (product release in this case) is ratelimiting (31, 32). We investigated reactions of the mammalian θ-class GST enzymes GST 5-5 (rat) and T1-1 (human), in light of the findings with GST DM11, and fit the data with a kinetic model. Collectively, the results indicate that the mammalian GSTs do not show burst kinetics and that the most likely rate-limiting step is the attack of the GSH thiolate on the haloalkane, which itself has little affinity for the enzyme. The relevance of these findings is discussed.

Experimental Procedures Chemicals. CH2Br2 and CH2Cl2 were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. These dihalomethanes were shaken with H2O to yield saturated aqueous solutions, which were used as stocks for enzymatic reactions (67 mM CH2Br2 and 203 mM CH2Cl2) (21). [Glycine-3H]GSH was purchased from New England NuclearDuPont (Boston, MA). Assays. All GST assays were done at 23 °C in 0.10 M potassium 3-(N-morpholino)propanesulfonate (pH 7.6) buffer. HCHO was usually measured using a modified Nash assay (33, 34) as modified and described elsewhere (21). In some experiments, a more sensitive assay was used, in which HCHO was reacted with 2,4-dinitrophenylhydrazine and the resulting hydrazone was analyzed by HPLC, with detection at 360 nm (35, 36). In some cases, a coupled assay was utilized for detection of HCHO, with Pseudomonas putida HCHO dehydrogenase and NAD+ reduction (21). The reaction of C2H5Cl with GST was analyzed (HPLC) as described previously (30). UV spectral assays were done with either a Cary 14-OLIS spectrophotometer or an OLIS RSM-1000 stopped-flow spectrophotometer (OLIS, Bogart, GA). Enzymes. GSTs 5-5 and T1-1 (both with C-terminal (His)5 tags) were expressed in Escherichia coli and purified as described (30) using the modifications reported recently.2 Protein concentrations were estimated using a bicinchoninic acid method (Pierce, Rockford, IL). Kinetic Models. Fits of steady state kinetic data points to estimate kcat, Km, and kcat/Km were done using Graphpad Prism (Graphpad, San Diego, CA). Fitting to kinetic models was done using the program DynaFit (37), which is available upon request (http://www.biokin.com; [email protected]). Programs were run on a Macintosh G4 computer (Apple Computer, Cupertino, CA) using Operating System X. Examples of files used in this work, with program scripts, are presented in the Supporting Information.

Figure 1. Steady state kinetics of GST reactions. (A) Dependence rates of conjugation of CH2Br2 (b) and CH2Cl2 (0) by GST 5-5 on their concentrations. (B) Dependence of rates of conjugation of CH2Br2 (b) and CH2Cl2 (0) by GST T1-1 on their concentrations. The GSH concentration was 7.5 mM.

Results Steady State Reactions. All reactions were done at 23 °C instead of the usual higher temperatures (21) in order to facilitate comparisons with other experiments intended to detect possible reaction bursts. GST 5-5 showed a hyperbolic dependence on GSH concentration, with a Km of 0.14 mM (Supporting Information). At a fixed GSH concentration (7.5 mM, approximating that in mammalian liver), typical nonhyperbolic plots of reaction velocity vs CH2Cl2 or CH2Br2 concentration were obtained (Figure 1), similar to others published previously (21, 30). GST 5-5 was more active than GST T1-1, and with both enzymes, the activity was higher with CH2Br2 than CH2Cl2, as expected from previous work.3 Examination of Possibility of Burst Kinetics. Recently, a kinetic burst has been observed in the reaction of the bacterial GST DM11 with C2H5Br (9). This result is only consistent with the existence of a rate3 In a previous paper (21), a hyperbolic plot of activity vs CH Br 2 2 concentration was observed, in contrast to the general patterns of GSTs 5-5 and T1-1 with all dihalomethanes and other haloalkanes. The reason for that reported result is unclear. A linear plot was observed here (Figure 1) and also at 37 °C (results not shown).

θ-Class GSH Conjugation Kinetics

Figure 2. Time dependence of conjugation of C2H5Cl with GSH by GST 5-5. The concentration of GST 5-5 was 7 µM, and the buffer used in these experiments was 0.10 M sodium borate (pH 8.0). Concentrations of [glycine-3H]GSH (14.7 mCi mmol-1) and C2H5Cl were 1.0 and 5 mM, respectively. (A) Reaction initiated with C2H5Cl. (B) Reaction initiated with GSH. Results presented are from single experiments (each point). The two parts (A, B) were done at different times with different GST 5-5 preparations and are not intended to be compared directly with regard to rates.

limiting step following product formation (31, 32). The possibility that a burst also occurs with the mammalian GSTs 5-5 and T1-1 was also considered. The first experiments were done with 1,2-epoxy-3-(4′nitrophenoxy)propane, which yields a spectral change upon conjugation with GSH (18, 38). No burst was detected in the spectral assays (results not shown), although the low extinction coefficient (∆360 ) 500 M-1 cm-1) might have precluded detection. The GST-catalyzed reaction of C2H5Cl yields a stable product, S-ethylGSH, which can be analyzed by HPLC (30). The GSH conjugation of C2H5Cl or C2H5Br by GSTs 5-5 and T1-1 is characterized by linear plots of activity vs haloethane (30), similar to those shown for dihalomethanes in Figure 1. This assay was utilized in the characterization of the burst in the work with GST DM11 (9). However, the reaction of GSH-complexed GST 5-5 with C2H5Cl did not yield a detectable burst (Figure 2A) nor did the reaction of a mixture of GST 5-5/C2H5Cl vs GSH (Figure 2B). In both cases, the plot extrapolated through the origin of the graph, and the amount of burst that might not have been detected is estimated to be 1% v/v). The difference between linearity and slight hyperbolic character is not easy to discern by graphing either. The remainder of this paper will utilize the view that there is a low but finite affinity (Kd of 0.1 M; Table 1) for the methylene dihalides, in part because the assumption is made that the reaction with the GS- is within the GST.

Discussion Linear kinetics of GSH conjugation of CH2Cl2 have been observed in numerous in vitro experiments (13, 21) and also in vivo (4, 12). This behavior of the θ-class

θ-Class GSH Conjugation Kinetics

mammalian GSTs with CH2Cl2 is observed not only with CH2Cl2 but also with most other dihalomethanes, vicdihaloethanes, and monohaloalkanes as well (21, 22, 30). Thus, the phenomenon is not the result of any peculiarities associated with the unstable products of the dihalomethane reaction (and regeneration of the substrate GSH; Scheme 1). In clear contrast, the reaction with the bacterial GST termed DM11 shows typical MichaelisMenten kinetics and a relatively low Km (21, 28, 30). The kinetic burst seen with GST DM11 (9) could explain the differences in steady state kinetic behavior relative to the mammalian GSTs, and we examined several pertinent features of the reactions with rat GST 5-5 and human GST T1-1, the θ-class enzymes relevant to metabolism of dihalomethanes and other small haloalkanes (23). Our results indicate the absence of burst kinetics with the mammalian GSTs and provide an explanation for the differential behavior with the bacterial GST DM11. The explanation is relevant to considerations of cancer risk assessment. Several experiments provided no evidence for burst kinetics, with extrapolation to the origin (t ) 0) in all cases examined, with the substrates C2H5Cl (Figure 2) and CH2Br2 and CH2Cl2 (Figure 3), in contrast to the sharp burst seen for GST DM11 (9). The kinetic modeling (Figure 4 and Table 1) was done with low affinity of GSTs for the product (GSCH2X), i.e., a Kd similar to that for GSH. In a simple equilibrium system, this step (4) should yield a rapid rate of product release (∼8 × 105 min-1; i.e., Kd ) k4/k-4 in this case). The koff rate (k4) may not be this rapid, and if k3 is low, a burst might not be detected. For instance, a koff of ∼8 min-1 was measured for GST DM11 (9) and this might not yield burst kinetics in the slow GST T1-1 reactions. However, in the GST 5-5 conjugation of CH2Br2, the estimated value of k3 was 240 min-1 and the turnover number (at 30 mM CH2Br2) was 30 min-1 (Figure 1A). If k4 were as low as 8 min-1, it would have been detected in the burst experiment (Figure 3A) (and the value would be impossible considering the results in Figure 1A); kinetic modeling with k4 ) 8 min-1 (Table 1) and adjusting k3 (even to a value >108 min-1) yielded kcat ∼4 min-1 (and a Km < 0.1 mM), clearly incompatible with experimental data. The point should be emphasized that this is a minimal model and the reaction contains more steps, which are pooled within some of the individual rate constants. Other solutions may be possible (also note that there is considerable flexibility in the rapid steps, as long as they are not so low as to affect the fits). However, this model explains (i) the Km for GSH (yielding 110 µM, cf. 140 µM experimentally for GST 5-5, Supporting Information), (ii) the lack of burst kinetics, and (iii) the differences in the rates with CH2Br2 and CH2Cl2 for both GST 5-5 and GST T1-1, without changing any rate constants except the displacement of the halide to form the product. The kinetic modeling suggests poor affinity of all of the GSTs for the alkyl halides in general. However, the possibility can be considered that the low Km of GST DM11 could simply be attributed to better inherent affinity of this enzyme for the haloalkanes. Although we do not have specific evidence against this possibility, there are some problems with the hypothesis: (i) GST DM11 would have to have a much higher affinity for all short chain haloalkanes, including the ethyl and methyl mono- and dihaloalkanes examined previously (21, 30) and (ii) the observed halide order on Km (21, 30) could

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Figure 5. (A) DynaFit simulation of GST DM11 results for C2H5X conjugation using the GST 5-5/T1-1 kinetic model and the rate constants shown in Table 1, adapted in part from Stourman et al. (9) (25 °C). The three traces were obtained with varying k3 values. [GSH] ) 2 mM in all cases. (B) Experimental results for conjugation of C2H5Br and C2H5Cl by GST DM11 at 37 °C (from ref 30). In part A, the following parameters were calculated with the rate constants presented in Table 1. For k3 ) 3.6 × 104 min-1: kcat, 4.1 min-1; Km, 0.28 mM; kcat/Km, 14.6 min-1 mM-1. For k3 ) 6 × 103 min-1: kcat, 4.6 min-1; Km, 1.5 mM; kcat/Km, 1.5 min-1 mM-1. For k3 ) 103 min-1, kcat/Km ) 0.12 min-1 mM-1.

only be explained by differential affinities between chlorides and bromides. The halide order, i.e., the differences in reactions with Br and Cl in this case, can be compared to kinetic isotope effects (31, 39) or elemental oxygen/sulfur effects on phosphate reactions (40) in that the major difference between pairs of substrates is the strength of the carbonhalogen bond, if we assume no large effect of the halide on enzyme-substrate binding or positioning. The placement of a rate-limiting step for product release (or any step following product release) has the effect of making the Km much lower than the true Kd. Previous examples are the P450 2E1-catalyzed oxidation of ethanol and acetaldehyde (36, 41) and microsomal epoxide hydrolase (42). In such situations, the effect of protium substitution with a heavy isotope (e.g., deuterium) is to obtain a kinetic deuterium isotope effect on Km but not kcat (36, 39). If the model for GST DM11 is correct (Figure 5), then the halide effect on Km can be explained in a similar way. The model yields changes in Km with only variation in k3, which would be the result of changing the ease of bond breaking. k3 is not the rate-limiting step, so kcat is unaffected. However, Km and thus kcat/Km are readily altered (Figure 5A). In our experience here and previously (21, 30), (rat) GST 5-5 has rather consistently been a better catalyst of all alkyl halide conjugations than (human) GST T1-1.

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The exact reason is not clear, but the modeling (Table 1 and Figure 4) is consistent with a difference in k3. All fits are acceptable if the other rate constants are used for both enzymes (Figure 4). The difference between k3 for the conjugation of CH2Br2 and CH2Cl2 is similar (10fold) for both enzymes, consistent with the view that a difference in another step (e.g., GSH deprotonation) is not the issue.4 With GST DM11, the rate of product release is slow, as clearly demonstrated by the burst kinetics (9). However, some product (GSCH2X) must escape the enzyme, because expression of GST DM11 in Salmonella typhimurium increases the mutagenicity of dihalomethanes (21, 43) (and also dihaloethanes (30)). The measured koff (k4) of 8 min-1 for GST DM11 (9) at 23 °C is similar to the turnover number of GSTs 5-5 and T1-1 at low dihalomethane concentrations (21). Indeed, a reexamination of the results of Wheeler et al. (21) in the context of further work indicates that at lower dihalomethane concentrations there is a clear pattern of increased mutagenicity of CH2Br2 > CH2Cl2 for the activation of these compounds by GST DM11, as seen with GSTs 5-5 and T1-1 (21). The number of mutants seen at lower dihalomethane concentrations with GST DM11 is similar to the number seen at higher substrate concentrations with GSTs 5-5 and T1-1 (21). Clearly the enzyme parameter of choice in making comparisons is the enzyme efficiency, kcat/Km (this is generally true in most situations). In no case is the change in Km necessarily due to altered substrate affinity, and we make a case that changes in the rate of carbonhalogen bond cleavage alter Km (Figure 5). Why GST DM11 holds GSCH2X (or GSC2H5) tightly (9) is not clear. A high koff (k4) and diffusion-limited transfer (GSTs 5-5 and T1-1) allow for reaction with DNA, at least in solution. Furthermore, recent work in this laboratory has shown that at high dihalomethane concentrations, GSTs DM11, 5-5, and T1-1 can all generate products from CH2Br2 and CH2Cl2 that will modify nucleosides to form GSCH2-containing adducts.2 Exactly how well the products move to DNA within cells is unknown. One of the problems is the instability of the derived DNA adducts (44, 45),2 making adduct analysis technically difficult. However, damage to Methylobacterium dichloromethanicum cellular DNA resulting from GST DM11-mediated damage from CH2Cl2 has been demonstrated by Kayser and Vuilleumier (45). With regard to the relevance of in vitro results to physiologically based pharmacokinetic in vivo models (for rodents and humans) and risk assessment, the in vitro parameter for use is clearly the enzyme efficiency, kcat/ Km. It should be emphasized that this parameter varies considerably among individual humans because of the polymorphism (gene deletion) of GST T1-1 (46), as opposed to variability due to induction or inhibiton. We have consistently found lower rates of CH2Cl2 conjugation for human GST T1-1 than for rat GST 5-5 in this work (Figure 1) and previous studies (20, 21, 30). The rates in human cytosols are also less than in rat cytosol (13). Mice have even higher rates of CH2Cl2 conjugation than rats (13), due to the presence of a θ-class GST with higher activity (24). Mainwaring et al. (47) have also provided evidence for a nuclear localization in the mouse (47). With all haloalkanes, including dihalomethanes, the effect of a Br constituent is to increase the rate of reaction ∼10fold > Cl (Figure 1 and refs 21 and 30), when this is the

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site of initial attack. Mixed halides, e.g., CH2BrCl, give varying results in different assays, probably depending upon the nature of the subsequent reaction. For instance, with CH2BrCl, the product is GSCH2Cl, which will readily hydrolyze to HCHO (regardless of whether Br or Cl is present), but GSCH2Cl might have different properties than GSCH2Br in reacting with DNA. The details of these differences are still not particularly clear, due to the difficulty in studying the reactive GSCH2X compounds (8-11, 44).2 However, we have shown that GSCH2CH2Cl is more mutagenic than GSCH2CH2Br when added to S. typhimurium cells (21), probably because of stability (GSCH2H2F is even more mutagenic). In conclusion, we still do not have all of the desired mechanistic information relevant to GSH-dihalomethane reactions, due to the difficulties in working with the DNA adducts (44, 45).2 Clearly, CH2Cl2 can be activated to a mutagen, without an obligate role for HCHO. Our studies presented here provide some insight into issues regarding GST-catalyzed conjugation of dihalomethanes and show some of the differences between GSTs DM11, 5-5, and T1-1, explaining some of the behavior. The driving force for the differences toward individual haloalkanes appears to be the differences in rates of halide displacement, which are manifested in the parameter kcat/Km. Other features of the reactions are disguised by changes in other steps.

Acknowledgment. This work was supported in part by U.S. Public Health Service (USPHS) Grants R01 ES10546 and P30 ES00267 (F.P.G.). J.B.W. was the recipient of a USPHS postdoctoral fellowship F32 CA80376. We thank N. V. Stourman and R. N. Armstrong for communicating the information in reference 9 and other helpful discussions. Supporting Information Available: Determination of Km of GST 5-5 for GSH and sample DynaFit files and results for fitting Figure 4A and the simulation in Figure 5A. This material is available free of charge via the Internet at http://pubs.acs.org.

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