Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden
1 To whom correspondence should be addressed. E-mail: bengt.mannervik{at}biokemi.uu.se
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Abstract |
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Keywords: epoxide/glutathione transferase/selectivity/stilbene oxide/styrene oxide
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Introduction |
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Epoxides are important toxic agents found ubiquitously in the environment. They are commonly formed in biological oxygen metabolism, but can also derive from anthropogenic sources and are important synthons in the chemical industry owing to the versatility of the oxirane function. Some epoxides are highly reactive as a result of the polarized carbons of their strained three-membered ring system. However, most are relatively stable under physiological conditions. Many epoxides are well-known carcinogens, such as arene oxides of polycyclic aromatic hydrocarbons and aflatoxin B1, and they commonly occur as stereoisomers with different toxicities (Buening et al., 1978; Guengerich et al., 1996
). Epoxides can be detoxified in several ways, such as by addition of water catalyzed by epoxide hydrolases (Morisseau and Hammock, 2005
) or by addition of GSH catalyzed by GSTs (Boyland and Williams, 1965
; Hayakawa et al., 1975
). Some members of the Mu class GSTs are known to be particularly active with epoxides, especially the human GST M1-1, which possesses a uniquely high activity with the proestrogenic compound trans-stilbene oxide (tSBO) (Seidegård et al., 1988
). GST M1-1 is also the main isoenzyme catalyzing GSH conjugation to styrene-7,8-oxide (SO) (Pacifici et al., 1987
), which is a metabolite of styrene but is also used as a synthon of fine chemicals. The homologous 84% sequence identical human GST M2-2 has an activity that is three orders of magnitude lower with tSBO and more than 40-fold lower with SO compared with GST M1-1 (Ivarsson et al., 2003
).
The functional versatility of GSTs has evolved through multiple gene duplications, accompanied by divergent evolution of the homologs (Sheehan et al., 2001). In the Mu class there is evidence that a limited number of hypervariable residues drive the evolution towards novel enzyme functions (Ivarsson et al., 2003
). Residue 210 is one of the limited number of hypervariable positions in the Mu class GSTs. This is an active-site component, which in the five human class members is alternatively Ser (GST M1-1), Thr (GSTs M2-2 and M4-4), Asn (GST M3-3) or Gly (GST M5-5). We have previously discovered that a Ser210Thr interchange between GSTs M1-1 and M2-2 is largely responsible for the 1000-fold difference in their catalytic activities with tSBO (Ivarsson et al., 2003
). Here we further explored the importance of the active-site residue 210 for the enzymatic activity of these two Mu class GSTs by examining six variants with Ala, Ser or Thr in the scaffolds of GST M1-1 and M2-2, respectively. Selective effects on the regio- and stereoselectivities with enantiomeric epoxide substrates were demonstrated by these minimal alterations of the amino acid side chains. The epoxides included the enantiomers of tSBO and its cis-isomer (cSBO) and the enantiomers of SO. The marked effects of the mutations on the activity with epoxides stand in contrast to the minor activity changes with a number of alternative substrates. Structural modifications of residue 210 can clearly differentially alter the substrate-activity profile, such that the turnover numbers of certain electrophiles change by several orders of magnitude, whereas those of alternative electrophiles remain largely unchanged.
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Materials and methods |
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1-Chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid and reduced GSH were purchased from Sigma (St. Louis, MO) and 5-androstene-3,17-dione (
5-AD) from Steraloids (Newport, RI). 2-Cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG) was a generous gift from Dr David E.Jensen (Thomas Jefferson University, Philadelphia, PA). tSBO, cSBO and SO were obtained from Aldrich (Milwaukee, WI). Enantiomerically pure (R,R)- and (S,S)-tSBO were a kind gift from Dr Per I.Arvidsson (Department of Chemistry, Uppsala University, Uppsala, Sweden). Oligonucleotides were purchased from Thermo Electron (Ulm, Germany). Pfu DNA polymerase and Escherichia coli XL1 Blue cells were obtained from Stratagene (La Jolla, CA).
Construction of alanine mutants
Alanine mutants of GST M1-1 and M2-2 (M1Ala and M2Ala, respectively) were constructed through inverted polymerase chain reactions using Pfu DNA polymerase and custom-synthesized 5'-phosphorylated oligonucleotide primers. M1Ala was constructed using the pGEtacM1b-11 high-level-expressing clone of GST M1-1 (Widersten et al., 1996
) as the template, with the sense primer 5'-CCA AGA CCT GTG TTC GCT AAG ATG G-3' and the antisense primer 5'-GAG GAA GCG GCT GGA CTT C-3'. M2Ala was constructed using the forward primer 5'-CCA AGA CCT GTG TTC GCG AAG ATG-3' and reverse primer 5'-GAG GAA GCG GCT GGA CTT C-3', with the high-level-expressing clone pKHXhGM2 of GST M2-2 as the template (Johansson et al., 1999
). The PCR products were purified on agarose gel and blunt-end ligated. E.coli XL1 Blue cells were transformed with the ligation mixtures by electroporation and the mutations were verified by DNA sequencing. M1Thr (Hansson et al., 1999
) and M2Ser (Ivarsson et al., 2003
) were available from previous studies.
Expression and purification
Proteins were expressed and purified as described previously (Johansson et al., 1999) and the purity of the enzyme samples was confirmed by SDSPAGE with Coomassie Brilliant Blue staining. Protein concentrations were determined by absorbance measurements at 280 nm. The extinction coefficient used for the GST M1-1 variants was 39 000 M1 cm1 with a subunit molecular mass of 25.7 kDa. For the variant GST M2-2 enzymes, an extinction coefficient of 40 800 M1cm1 and a subunit molecular mass of 25.6 kDa were used.
Enzyme activity assays
Specific activities with CDNB, cyanoDMNG, ethacrynic acid and 5-AD were determined under standard conditions (Mannervik and Widersten 1995
). To investigate the effect of the mutations of residue 210 on the catalytic efficiency and stereospecificity, alternative epoxide substrates were used. The activities with the isomers of tSBO and SO were determined as described previously (Ivarsson et al., 2003
). The specific activity with cSBO was monitored at 255 nm (
255 = 492 M1 cm1) in 250 mM TrisHCl, pH 7.2, using 250 mM cSBO and 10 mM GSH.
Steady-state kinetic analysis
Steady-state kinetics with (R,R)- and (S,S)-tSBO were performed as previously described (Ivarsson et al., 2003). The kinetic constants with the SOs were determined as described previously (Ivarsson et al., 2003
), but with 10 mM GSH and varying the SO concentration between 0.5 and 8 mM. The conjugation of cSBO was monitored under the above conditions, varying the concentration of cSBO between 1.5 and 125 mM. Steady-state kinetic parameters were determined by fitting the MichaelisMenten equation to the data points using Prism 2.0 (GraphPad Software, San Diego, CA). kcat values were expressed per protein subunit.
Viscosity effects
The viscosity dependence of the kinetic parameters of GST M1-1 and GST M2-2 with (R,R)- and (S,S)-tSBO and GST M1-1 and M1Ala with (R)- and (S)-SO was determined by steady-state kinetics in the presence of different concentrations of viscosogen, either sucrose or glycerol (032%, w/v). The principles of the method have been described elsewhere (Bazelyansky et al., 1986; Blacklow et al., 1988
; Guha et al., 1988
) and the values of relative viscosity used were based on published data (Guha et al., 1988
). The effects on the GST M2-2-catalyzed reactions with tSBO were determined in order to rule out direct perturbation of the enzyme. This is a proper control since GST M2-2 catalyzes the reaction very poorly and hence cannot be diffusion limited. A decrease in the kcat of GST M2-2 was observed with increasing glycerol concentration. The effect on kcat was compensated for by the factor C1 (C1 = kcat0/kcat for GST M2-2). After the mentioned corrections, no significant differences were observed between the results using sucrose or glycerol as viscosogen.
High-performance liquid chromatography (HPLC)
The HPLC procedure used to resolve the conjugation product formed in the enzyme-catalyzed reaction between cSBO and GSH was as reported by deSmidt et al. (1987), with the following modifications: Enzyme-catalyzed reactions (0.5 ml) were run for 30 min at 30°C in 0.1 M 3-(N-morpholino)propanesulfonic acid, pH 7, using 1 mM GSH, 0.5 mM cSBO and 10210 mg of enzyme. The reactions were quenched with 50 ml of 5 M acetic acid. The samples were filtered through an IC Acrodisc 13 mm syringe filter (0.2 mM) and stored at 20°C until the time of analysis. The samples were analyzed using LaChrom Elite from Merck Hitachi (Darmstadt, Germany). Chromatography was performed on a 25 cm x 4.6 mm i.d. Nucleosil 100-5 C18 column and eluted at 0.5 ml/min with 40% methanol in 0.1 M ammonium acetate, pH 4.0. The rat GST M2-2 catalyzes the GSH conjugation to cSBO with almost exclusive selectivity for the carbon of R absolute configuration, resulting in the formation of the (S,S)-1 product to 98% (deSmidt et al., 1987
). This was used to identify the (R,R)- and (S,S)-1 products.
The product distribution achieved in the reaction between GSH and SO using the different enzymes were analyzed by HPLC. The samples were prepared as described Dostal et al. (1988). Both racemic and enantiomerically pure SO were used as starting materials and the reactions were run to 1020% completion. The samples were filtered as described above and stored at 20°C until the time of analysis. The reversed-phase HPLC-analysis of the conjugation products between GSH and SO was based on the procedure developed by Hernandez et al. (1983)
, with the modification that the above column was used and the flow rate was 0.5 ml/min. The column was equilibrated with buffer A (25 mM Trisphosphate buffer, pH 7.0 and 25 mM sodium sulfate) and the sample was injected followed by a 10 min washing step with buffer A. The GSH conjugates were then eluted with buffer B [buffer A containing 2.5% (v/v) methanol]. An additional washing step [20 min, 20% (v/v) methanol, 0.75 ml/min] was introduced between each injection. The products eluted after 95130 min and were detected at A254. Duplicate analyses were made from each sample.
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Results |
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Two mutants were constructed from each of the human wild-type enzymes GST M1-1 and GST M2-2. In GST M1-1, Ser210 was replaced by Ala or Thr resulting in M1Ala and M1Thr, respectively (Figure 1). The corresponding mutations in GST M2-2 were replacements of Thr210 by Ala or Ser (M2Ala and M2Ser, respectively). The resulting enzymes, and also the wild-type GST M1-1 and GST M2-2, were successfully expressed and purified. Specific activities were determined with a number of electrophilic substrates (Table I). Steady-state kinetic parameters were determined with different epoxide substrates (Table II). The substrate dependence of the rate in the reactions with tSBO did not reach saturation within the soluble range of the substrate and the kcat and KM values obtained with this substrate should be considered as crude estimates. However, the catalytic efficiencies (kcat/KM) could still be determined with adequate precision from the gradient at the origin of the rate-saturation curves.
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In contrast, remarkably enhanced catalytic activities with epoxides were observed when Thr210 in GST M2-2 was replaced by either Ala or Ser, generally resulting from an increase in the turnover number (kcat). The highest increase in catalytic efficiency (320-fold) was obtained with the M2Ser variant acting on the R,R-isomer of tSBO, the effect resulting from a 760-fold increase in the turnover number. Despite this dramatic improvement in catalytic activity, the reactions catalyzed by M2Ser and M2Ala do not reach the catalytic efficiency of wild-type GST M1-1, which still has more than 20 times higher efficiency with (R,R)-tSBO. In the GST M1-1 scaffold, both the M1Ala and M1Thr substitutions resulted in a marked decrease in catalytic efficiency with (R,R)-tSBO (20- and 600-fold, respectively), also mainly a kcat effect. With (S,S)-tSBO, the effects on the turnover numbers were less pronounced, whereas significant increases in KM values were observed.
All the enzyme variants displayed low activities with the cis-isomer of SBO. GST M2-2 had no detectable activity (<0.5 nmol/min/mg protein) with cSBO, whereas both M2Ala and M2Ser demonstrated measurable activities with this substrate (Table I). No analysis of the steady-state kinetics was performed with the GST M2-2 variants owing to the low activities. The values of the catalytic efficiencies of the GST M1-1 variants are similar, even though differences between the enzymes were observed for both kcat and KM; lower kcat values were largely compensated by lower KM values.
With the smaller epoxide substrate SO, the effects of the mutations were less pronounced. The low catalytic efficiency of wild-type GST M2-2 is improved 20-fold with (R)-SO and 80-fold with (S)-SO, by both the M2Ser and M2Ala substitutions. Compared with GST M1-1, these variants have similar catalytic efficiencies with SO. The M2Ser variant actually has a higher kcat than wild-type GST M1-1, but the efficiency is lowered by the high KM value. Surprisingly, the M1Ala replacement confers a significant increase in turnover number with (S)-SO, making the M1Ala enzyme the most efficient variant with this substrate.
Stereoselectivity of the GSTs acting on enantiomeric substrates
The degree to which an enzyme discriminates between two enantiomeric forms of a substrate can be estimated from the ratio of specificity constants [(kcat/KM)A/(kcat/KM)B] for the two enantiomers (the E-value). An E-value <10 indicates low selectivity, a value >20 indicates moderate selectivity possibly useful for applications, whereas E > 100 indicates excellent selectivity. The GST variants investigated are, to varying degrees, selective for (R,R)- over (S,S)-tSBO, with E-values ranging from 2.7 to 58 (Table II). Most of the enzymes display fairly poor selectivity between the two enantiomers of tSBO, but the M2Ser mutant has an E-value of 58. A Ser210 appears to be favorable for both enantioselectivity and catalytic activity (kcat). For resolution of a mixture of the (R,R)- and (S,S)-tSBO enantiomers, M2Ser would be the most useful catalyst of the variants investigated.
The enantioselectivities between the enantiomers of SO are low and, in contrast to the preference for the R-configured carbon of tSBO, most of the enzyme variants have a preference for the S-enantiomer of SO (Table II). The Ala substitution slightly improved the enantioselectivity in both GST M1-1 and GST M2-2 and the M1Thr replacement resulted in a reversed stereoselectivity.
Transition state stabilization
The enzyme-catalyzed reactions with chiral compounds may proceed via alternative transition states (TS) with different free energies for the enantiomers (Figure 2). The differences in binding energy [Gb = RT ln(kcat/KM)A/(kcat/KM)B] with the enantiomers of tSBO [
] were estimated to be in the range 0.62.4 kcal/mol for the GST variants; the largest separations were obtained with M2Ser and M1Ser, 2.4 and 1.8 kcal/mol, respectively. This
Gb is energetically of the size expected for a hydrogen-bond interaction (15 kcal/mol) (Fersht, 1999
).
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Regioselectivity
The reaction between GSH and tSBO produces a single product from each enantiomer (Figure 2A). In contrast, the two carbons of the oxirane function of cSBO and SO are stereochemically distinct (Figure 2B and C) and, depending on the carbon that GSH is attacking, two regioisomeric products are formed, which can be separated by HPLC (Figure 3). All enzyme variants were shown to possess high positional selectivity for the carbon with R absolute configuration in cSBO, yielding almost exclusively the (S,S)-1 product (Table III). In the reaction between racemic SO and GSH, four different products can be formed, two from each enantiomer (Figure 2C). Most GSTs variants showed a preference for the benzylic carbon of SO and a selectivity for the S-enantiomer, consistent with the values from the kinetic experiments (Table IV). In contrast to all other enzyme variants, M1Thr displayed a preference for the distal rather than the benzylic carbon of (S)-SO and a reversed enantioselectivity (Figure 2C and Table IV).
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The rate of an enzyme-catalyzed reaction can be limited by either the chemical transformations of the reactants or diffusion-dependent steps such as formation of the encounter complex, product release or conformational changes of the protein. The second-order rate constants (kcat/KM) of the GST variants with the epoxide substrates are considerably lower than the predicted second-order rate constants for diffusion-limited formation of encounter complex (109 s1 M1). Therefore, substrate binding is not limiting the rate of the enzymatic reaction. The viscosity dependence of the turnover number reveals the extent to which product release is rate limiting. The turnover number obtained in the absence of viscosogen (
) is related to the value obtained at a higher viscosity (
). If product release is fully rate limiting, it is expected that a plot of
versus the relative viscosity will have a unit slope.
A significant decrease in kcat was observed upon addition of viscosogen in the GST M1-1-catalyzed reaction with (R,R)-tSBO (Figure 4A and B), indicating that product release is partially rate limiting. The slopes of the versus relative viscosity plots were found to be 0.64 ± 0.02 and 0.69 ± 0.09 using sucrose and glycerol as viscosogens, respectively. In contrast, no GST variant showed a viscosity effect with (S,S)-tSBO (data not shown), indicating that chemistry is fully rate limiting with this substrate.
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Discussion |
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The significance of residue 210 in the reactions catalyzed by Mu class GSTs has been discussed (Johnson et al., 1993; Shan and Armstrong 1994
; Codreanu et al., 2002
; Ridder et al., 2002
; Ivarsson et al., 2003
). It has been speculated that an interaction between the hydroxyl groups of residue 210 and Tyr116 activates Tyr116 as an electrophile and that a direct or water-mediated, hydrogen bond from residue 210 to the electrophilic substrate increases the catalytic efficiency. In the present study, the functional importance of this active-site residue was probed by small structural variations through interconversion of Ala, Ser and Thr in the GST M1-1 and GST M2-2 scaffolds (Figure 1). To analyze the data we assign Ser210 in both the GST M1-1 and M2-2 scaffolds as starting points and examine the consequences of elimination of the hydroxyl group from or addition of a methyl group to this residue. Eliminating the hydrogen-bonding ability of the residue by the Ala210 replacement has only limited effects on the enzymatic activities with several of the investigated epoxide substrates. A marked decrease in specific activity in both the GST M1-1 and M2-2 scaffolds is observed only with CDNB and (R,R)-tSBO, which demonstrates that a hydroxyl group at position 210 is not necessary for the catalytic activity. The most detrimental effect is found in the isomerization of
5-AD catalyzed by GST M1-1, which suffers a 20-fold decrease in activity as a consequence of the substitution. When Ser210 instead is provided with an additional methyl group at the ß-carbon, there is a great impact on the ability of the enzymes to catalyze the reaction between GSH and the various epoxide substrates, indicating that steric hindrance by the methyl substituent of Thr210 interferes with catalysis.
It is noteworthy that some of the catalytic activities (i.e. the low activity with ethacrynic acid and the high activity of GST M2-2 with cyanoDMNG) are not at all affected by the interchange of residue 210 between Ala, Ser and Thr, indicating that other residues determine the catalytic parameters.
Mechanism with epoxide substrates
In the GST-catalyzed reaction with epoxides, the oxirane ring is oriented for a nucleophilic attack on one of the carbon atoms along a trajectory projecting through the carbon and oxygen atoms. The approach of the deprotonated sulfhydryl group of GSH results in opening of the strained ring structure and an inversion of the configuration of the carbon (Figure 5). During the oxirane ring opening, the nascent oxyanion receives electrophilic assistance through a hydrogen bond donated by Tyr116, as evidenced by the crystal structure of the homologous rat GST M1-1 in complex with the product obtained by conjugation of GSH and phenanthrene-7,8-oxide (Ji et al., 1994). In this structure, a hydrogen bond is also donated by Ser210 to a lone electron pair of the phenolic oxygen of Tyr116. A hydrogen-bond interaction between Tyr116 and the main chain amide of residue 210 has also been suggested (Johnson et al., 1993
).
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The rate-limiting step
To understand fully the influence of a mutation on the catalytic activity of an enzyme, the rate-limiting step in catalysis needs to be established. An effect of a mutation on the chemical mechanism may be masked if the rate-limiting step is not chemical in nature, but rather a physical step such as substrate binding, product release or a conformational transition in the enzyme. It has been suggested previously that the GST M2-2-catalyzed conjugation of CDNB is limited, at least partly, by product release (McCallum et al., 2000). A slow conformational change of the Mu loop, involved in gating the access to and the egress from the active site, was proposed to limit catalytic turnover. The denitrosation of cyanoDMNG catalyzed by GST M2-2, which is a reaction with fairly high efficiency, is also partly limited by product release (unpublished data of the present authors). Furthermore, segmental motion associated with rate-limiting product release has been detected in the homologous rat GST M2-2 (Codreanu et al., 2002
). Surprisingly, the relatively slow GST M1-1-catalyzed reaction with (R,R)-tSBO displays a fractional viscosity dependence consistent with a rate-limiting product release (Figure 4). A contribution of Ser210 to the rate of the chemical step would therefore not be fully apparent in the observed turnover number. The reaction with (S)-SO catalyzed by M1Ala displays a fractional viscosity dependence, whereas the GST M1-1-catalyzed reactions with SO and with (S,S)-tBSO show no such tendency. Consequently, to improve further the catalytic efficiency and selectivity with these substrates, the rates of both the chemical transformation and the product release need to be increased.
Enantioselectivity
The discrimination between competing substrates, such as enantiomers, is achieved by a combination of electrostatic and steric effects contributing to productive substrate binding and stabilization of the TS along the reaction coordinates. The simple case is illustrated by the enantiomers of tSBO, where each enantiomer produces a single separate product in reactions proceeding through TSs subjected to different stabilizing interactions (Figure 2A). All the investigated Mu class variants display, to varying extents, a preference for the R,R-enantiomer of tSBO with differences in TS stabilization [] corresponding to 0.62.4 kcal/mol (Figure 2A). This indicates that amino acid side chains other than residue 210 are involved in providing the enzyme with this shared selectivity. The low activity with the S,S-enantiomer might be ascribed to steric clashes of the substrate with different structural elements in the H-site. The selectivity for the R-configured carbon of epoxide substrates is in line with the previously reported selectivities of GST M1-1 with a number of polycyclic aromatic hydrocarbons (Sundberg et al., 1997
).
However, it is noteworthy that a Ser210 in the scaffolds of both GST M1-1 and GST M2-2 increases the selectivity by 7- and 10-fold with the enantiomers of tSBO, compared with the selectivity of the Ala210 variants. The higher enantioselectivity corresponds to an enhanced TS stabilization ( and
) of 1.8 and 0.9 kcal/mol for the GST M1-1 and M2-2 variants, respectively, which is in the lower range for a hydrogen bond. Since the reaction catalyzed by wild-type GST M1-1 (M1Ser) is partly limited by product release, the full potential of this stabilizing interaction cannot be realized in the rate of the reaction. It is possible that the considerably higher enantioselectivity provided by a Ser210 is due to a direct hydrogen bond between the hydroxyl group of Ser210 and the oxirane oxygen of (R,R)-tSBO, but not with the oxygen of (S,S)-tSBO.
Regioselectivity with cSBO
Next, the regioselectivity with the pro-chiral compound cSBO is considered (Figure 2B). The positional selectivity with this compound is probably largely determined by how cSBO is accommodated in the active site and the location of the two carbons of the oxirane function relative the thiolate anion of GSH. The differences in electrostatic and steric effects are manifested in the pronounced predominance of the (S,S)-1 product formed from attack on the R-configured carbon (Figure 2B and Table III). This marked regio-preference is largely unaffected by the substitutions of residue 210 in the GST variants. The regioselectivity of the R-configured carbons of cSBO also matches the previously reported GST M1-1 selectivity for the R-configured carbon of the two arene oxides benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide (Dostal et al., 1988). Possibly a steric effect of residue 10 (isoleucine in both GST M1-1 and GST M2-2) contributes to this selectivity, as described previously for the rat GSTs M1-1 and M2-2 (Shan and Armstrong, 1994
).
Regio- and enantioselectivity with SO
A more complex case is the enzyme-catalyzed GSH conjugation of racemic SO, in which both enantio- and regioselectivity must be taken into consideration (Figure 2C). The enantioselectivity is relatively poor, with only a slight preference for the S-enantiomer, contrasting with the previously described preference for R-configured carbons. Possibly, this small substrate can adopt different orientations in the active site owing to less interference from steric clashes with structural elements in the H-site (Figure 1). Alternative binding modes have been observed in the active site of human GST A1-1 with the substrate ethacrynic acid (Cameron et al., 1995). The regioselectivity is more pronounced than the enantioselectivity (Table IV and Figure 2C). Both the enantioselectivity and the positional discrimination are improved by an Ala210 substituent, indicating that Ala allows a different positioning of SO in the active site. Particularly high regioselectivity, combined with relatively high catalytic efficiency, is achieved by the Ala210 substituent in the GST M2-2 scaffold.
The stereo- and regioselectivities of the Mu class GSTs are accomplished in two phases in the catalysis. Starting from a racemic mixture of SO the enzyme discriminates between the enantiomers. Second, the enzyme selects either of the two carbons of the epoxide function leading to a partitioning between the two regioisomeric products. This biphasic discrimination is evident from the activities of the three enzyme variants M1Ala, M2Ala and M2Ser, which discriminate almost equally well between the two enantiomers of SO (Table II), but produce substantially different proportions of the alternative products (Table IV). Conformational transitions of the enzyme may be linked to the partitioning of the bound substrate between the alternative TSs that lead to the regioisomeric products.
Conclusions
The rational design of enzymes with a certain enantio- and regioselectivity would be of great value considering the growing interest for enzyme catalysis in the chemical industry. How to design enzymes rationally to meet requested selectivities is not obvious, since relatively minor alterations in the active site of the enzyme or in the structure of the substrate might have a very large impact on the outcome of the reaction, as demonstrated in this study.
GSTs are enzymes with high functional plasticity. The low catalytic activity with epoxides displayed by GST M2-2 is clearly not an important native function for this enzyme. Epoxide conjugation is drastically improved by mutation of residue 210 whereas other, higher, activities display robustness towards alterations of the residue. These findings are in line with the notion of evolvability of promiscuous protein functions with retained native activities recently presented by Tawfik and co-workers (Aharoni et al., 2005). The differential effects on the catalytic activities of Ala, Ser and Thr in position 210 provide further evidence of the importance of this residue on the functional diversity of the Mu class GSTs. In a previous study, we demonstrated that residue 210 is hypervariable among the Mu class GSTs (Ivarsson et al., 2003
) and this position may accordingly be instrumental in the evolution of nascent functions in the protein structure.
GST M1-1 was discovered as a polymorphic enzyme distinguishable from other GSTs by a different activity profile (Warholm et al., 1980, 1981
). Its activity with benzo(a)pyrene-4,5-oxide suggested an important role in the cellular protection against mutagenic and carcinogenic epoxides (Warholm et al., 1981
). It is clear that cells lacking GST M1-1 suffer more chromosome damage from epoxides than cells expressing the enzyme. Nevertheless, the significance of the genetic polymorphism is not obvious since the null genotype is frequently found in humans (Seidegård et al., 1988
). The absence of the gene may suggest the lack of selective advantage to a carrier of the gene. On the other hand, a GST M1-1 gene duplication has been reported (McLellan et al., 1997
), indicating the usefulness of the gene. A possible explanation is that GSTs not only catalyze the inactivation of toxic agents, but also produce more toxic agents from compounds, as exemplified by dihaloalkanes (Rannug et al., 1978
; Guengerich, 2003
). Variations among human populations of different ethnicities may therefore be linked to differential needs of detoxication capacity due to dietary or other environmental factors (Johansson and Mannervik, 2001
). The present investigation shows that alternative needs of GST functions can be met by point mutations, thereby providing facile adaptations to chemical challenges arising in the environment.
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Acknowledgements |
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Received August 18, 2005; accepted August 31, 2005.
Edited by Dan Tawfik