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Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA
Reprint requests to: Roberta F. Colman, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA; e-mail: rfcolman{at}chem.udel.edu; fax: (302) 831-6335.
(RECEIVED June 9, 2003; FINAL REVISION July 14, 2003; ACCEPTED July 14, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03249303.
1 The amino acid sequences of the human and porcine GST 
isozymes are 83% identical plus 6% similar, justifying the use of the crystal structures of the human enzyme to understand the experimental data of the porcine enzyme. 
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(GST ): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.
Keywords: Affinity labeling; glutathione S-transferase; GST ; monobromobimane
Abbreviations: GST, glutathione S-transferase • GST, class glutathione S-transferase • mBBr, monobromobimane • CDNB, 1-chloro-2,4-dinitrobenzene • GS-Bimane, S-(glutathionyl)-bimane • ANS, 8-anilino-1-naphthalene-sulfonate • BSP, bromosulfophthalein • mB-Cys, S-(cysteinyl)-bimane, NEM-Cys, S-(N-ethylsuccinimido)cysteine • PTH, phenylthiohydantoin • TFA, trifluoroacetic acid
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The cytosolic GSTs, which exist as either homo- or heterodimers, are currently divided into at least eight classes (named 
, 
, µ, 
, , 
, 
, and 
) on the basis of their physical and chemical properties (Mannervik and Danielson 1988; Tsuchida and Sato 1992; Board et al. 2000). Isozymes within a given class have about 86% identity in amino acid sequence. However, isozymes from different classes are quite dissimilar, having, for example, only about 29% amino acid sequence identity between and or µ classes. Although each isozyme generally exhibits broad substrate specificity, most have unique catalytic attributes that are important in defining the role of a particular isozyme in the metabolism of electrophiles (Armstrong 1991). Some classes of GSTs (especially ) have been shown to have distinct binding sites for compounds other than the primary xenobiotic substrate. These additional sites provide one way of extending the functions of a given glutathione S-transferase.
Among various GSTs, the -class (GST or GST P1-1) has attracted attention as a reliable preneoplastic or neoplastic marker enzyme because it is overexpressed in certain tumor cells (Park et al. 2001). Moreover, GST has been implicated in the development of resistance of tumors towards various anticancer drugs, such as Adriamycin, cisplatinum, melphalan, and Etoposode in resistant tumor cells (Aceto et al. 1989; Hasson et al. 1991; Kase et al. 1998; Morrow et el. 1998; Niitsu et al. 1998). Thus, the design of highly potent GST selective inhibitors can be useful in increasing the effectiveness of commonly used anti-cancer agents. The aim of this project is to use affinity labeling to explore the question of whether GST has more than one site for xenobiotic substrates and to compare the active site of GST with that of other classes of glutathione S-transferases to understand its distinctive features.
Monobromobimane (mBBr) is a nonfluorescent hydrophobic compound that reacts with nucleophiles to give fluorescent derivatives (Kosower and Kosower 1987). In previous studies, mBBr has been shown to act as an affinity label for and µ class GSTs (Hu and Colman 1995; Hu et al. 1996); however, GST , which is quite distinct from the other two classes, was not tested for its ability to react with mBBr. In this article, we report the results of the affinity labeling of pig lung GST using mBBr. The sites in GST labeled by mBBr are different from those modified in the and µ class GSTs. Covalent modification of GST by mBBr causes differential loss of activity toward two substrates (1-chloro-2,4-dinitrobenzene and mBBr), suggesting the existence of more than one binding site for xenobiotic substrates in the isozyme.
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class glutathione S-transferase (GST ); the reaction follows normal Michaelis-Menten kinetics. Figure 1
compares the structures of mBBr and 1-chloro-2,4 dinitrobenzene (CDNB), both substrates for GST . In GST , the apparent Km values for mBBr and CDNB were 33 ± 3 and 910 ± 40 µM, respectively, and the apparent Vmax values were 15 ± 1 and 57 ± 3 µmole/min/mg, respectively. Thus, the enzyme has a higher apparent affinity for mBBr, but a lower Vmax than for CDNB, the more traditional substrate for GST. For comparison, -class glutathione S-transferase (1-1) has a Km of 26 ± 3 µM and a Vmax of 87 ± 4 µmole/min/mg for mBBr as a substrate; while µ-class glutathione S-transferase (3-3) has a Km of 0.63 ± 0.06 µM and a Vmax of 3.5 ± 0.1 µmole/min/mg, when using mBBr as a substrate (Hu et al. 1996). The enzyme is distinctive, but is somewhat closer to class GST in its kinetic characteristics. S-(Hydroxyethyl)bimane, formed by reaction of mercaptoethanol and mBBr with displacement of the bromide, is structurally similar to mBBr (see Fig. 1), but cannot react with a nucleophile such as the —SH of glutathione. S-(Hydroxyethyl)bimane is not a substrate of GST , but rather functions as a competitive inhibitor with respect to mBBr as a substrate. This conclusion is based on the observation that addition of 50 µM or 100 µM increases the apparent Km for mBBr without changing the Vmax, yielding a KI of 56 ± 6 µM. When tested with respect to CDNB, the same concentrations of S-(hydroxyethyl)bimane did not act as an inhibitor, indicating that CDNB and mBBr occupy distinct substrate sites of GST .
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, it apparently binds to the enzyme. Therefore, mBBr was evaluated for its ability to react covalently with pig glutathione S-transferase . Incubation of GST with 0.15 mM mBBr at pH 7.0 and 25°C, in the absence of glutathione, results in a time-dependent inactivation of the enzyme (using mBBr as a substrate) with a rate constant of 0.066 min-1 (Fig. 2). Control enzyme, incubated under the same conditions but in the absence of the reagent, showed no change in activity during the same period.
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-class glutathione S-transferase by mBBr were tested to determine their effects on the rate constant for inactivation of GST (measured from the activity towards mBBr as a substrate). GST ligands were added to the reaction mixture at concentrations at least five times their reported Km or KI (Pettigrew et al. 2001) to determine their effects on the rate constant for inactivation of GST by 0.15 mM mBBr. In Table 1, the results are expressed as the ratio of the inactivation rate constant measured in the presence of a particular ligand (k+L) to the rate constant measured in the absence of any ligand (k-L).
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, lines 2–4). S-Methylglutathione is an alkyl derivative of glutathione, which binds within the glutathione site. With 5 mM S-methylglutathione added to the reaction mixture, the rate of inactivation is decreased twofold. S-Hexylglutathione is similar in structure to S-methylglutathione, except that the alkyl side chain is longer, allowing it to bind to an electrophilic substrate binding site. The S-hexylglutathione decreases the rate constant to 16% of that in the absence of ligands (Table 1, line 3). S-(p-Nitrobenzyl)glutathione is an aryl derivative of glutathione, which binds to both the glutathione active site and an electrophilic substrate binding site; complete protection against inactivation is provided (Table 1, line 4). Because S-methylglutathione exerts a much less protective effect than do S-hexylglutathione or S-(p-nitrobenzyl) glutathione, the reaction of mBBr cannot take place entirely within the glutathione site.
2,4-Dinitrophenol, a nonreactive analog of the electrophilic substrate CDNB, does not protect against inactivation by mBBr (Table 1, line 5). However, the addition of dinitrophenol and S-methylglutathione together protect the enzyme against inactivation by mBBr more than does either ligand alone (Table 1, line 6).
In contrast to dinitrophenol, S-(hydroxyethyl)bimane (as a nonreactive analog of the substrate monobromobimane) markedly decreases the inactivation rate (Table 1, lines 7 and 8) showing that S-(hydroxyethyl)bimane competes with mBBr in occupying the target site on GST . The magnitude of protection against inactivation by mBBr depends on the concentration of S-(hydroxyethyl)bimane, allowing the calculation of a KD of 59.6 µM for S-(hydroxyethyl)bimane from its ability to decrease the inactivation rate; this KD value is comparable to the KI for S-(hydroxyethyl)bimane determined from its behavior as a competitive inhibitor with respect to the substrate monobromobimane.
Glutathionyl-bimane (GS-Bimane) totally prevents inactivation of GST by mBBr (Table 1, line 9), and is thus a more effective protectant than S-(hydroxyethyl)bimane at the same concentration (330 µM). This protective effect indicates that the glutathione and mBBr sites in GST must not be far apart and that both sites must be occupied to completely protect it from inactivation.
In addition to its catalytic function, GST has a high binding capacity for a variety of nonsubstrate compounds (Bico et al. 1994). 8-Anilino-1-naphthalene-sulfonate (ANS) and bromosulfophthalein (BSP), shown in Figure 1, are two compounds known to bind to nonsubstrate binding sites in GST . BSP (KD = 1.1 µM) binds to GST more tightly than does ANS (KD = 14.1 µM; Bico et al. 1994); this previous study showed that ANS and BSP are noncompetitive inhibitors with respect to CDNB and glutathione, suggesting they bind to a different form of the enzyme and this may be at different sites. When tested, neither BSP nor ANS decreased the rate of inactivation of GST by mBBr (Table 1, lines 10 and 11). These results indicate that ANS and BSP do not bind in the same enzyme site targeted by mBBr.
Dependence of rate of inactivation on mBBr concentration
An affinity label characteristically forms a reversible enzyme–reagent complex prior to the irreversible modification (Colman 1997). The existence of a reversible enzyme–reagent complex is indicated by a "rate saturation effect" in which the rate of modification increases with increasing reagent concentration until the enzyme site is saturated with reagent. The observed rate constant, kobs, for mBBr inactivation of GST , was measured over a range of concentrations from 0.05 mM to 0.6 mM in the absence and presence of ligands. In the presence of 5 mM S-methylglutathione, kobs exhibits a hyperbolic dependence on the mBBr concentration with a KI of 127 ± 31 µM and a kmax of 0.058 ± 0.005 min-1 (Fig. 3A). These results suggest that mBBr binding occurs prior to enzyme inactivation in the presence of S-methylglutathione. In contrast, when the enzyme is incubated together with 330 µM S-(hydroxyethyl) bimane, the observed inactivation rate constant, kobs exhibits a linear dependence on [mBBr] with a second-order rate constant of 0.059 ± 0.008 min-1 mM-1 (Fig. 3B), suggesting either that there is no binding of mBBr prior to modification of the enzyme in the presence of S-(hydroxyethyl) bimane or that the affinity for mBBr is weak under these conditions (KI > 0.7 mM). In the absence of any ligand, the reaction once again exhibits a linear dependence on [mBBr] with a second-order rate constant of 0.44 ± 0.05 min-1 mM-1, showing that a bimolecular reaction of mBBr and the enzyme predominates in the absence of substrate analogs over the mBBr concentration range shown (Fig. 3C).
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was incubated with 0.15 mM mBBr in the absence or presence of added protectants. Subsequently, the modified enzymes were isolated and the incorporation of bimane was measured from its characteristic absorbance at 390 nm. The incorporation of mBBr into GST was measured as a function of time (0 to 30 min) in the absence of added ligands, and the reagent incorporation was plotted as a function of loss of activity at the same time (Fig. 4). Extrapolation to totally inactive enzyme yields about 2 mole reagent/mole subunit. These results suggest that inactivation results from modification of at most two amino acid residues.
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shows the effect of added ligands on the incorporation of mBBr into GST . Incubation of GST with 0.15 mM mBBr for 30 min affords a modified enzyme that is 87% inactivated and contains 1.7 mole of reagent/mole of subunit. Incubation under the same conditions, but with added GS-Bimane or S-(p-nitrobenzyl)glutathione leads to enzyme with no significant incorporation of mBBr and no loss of enzymatic activity, suggesting that the covalent reaction occurs in the region of the active site. The addition to the reaction mixture of either S-methylglutathione or S-(hydroxyethyl)bimane yields enzyme that retains appreciable activity and has decreased incorporation (Table 2). These results suggest that the mBBr reaction occurs at two locations—one in the glutathione binding region, and the other in the region of the xenobiotic substrate. Furthermore, when adding both S-(hydroxyethyl)bimane and S-methylglutathione to the 30-min inactivation mixture, an enzyme is obtained with almost no inactivation and an incorporation of only 0.2 mole mBBr/mole subunit; this minor incorporation is probably not at the active site of GST .
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compares the percent inactivation as a function of time of incubation with mBBr, using as substrates either CDNB or mBBr. The residual activity is different in the two assays at each time. For example, modification of GST by 0.15 mM mBBr for 30 min yields an enzyme with only 13% residual activity when assayed with mBBr as substrate, but with 39% residual activity for CDNB as substrate. These results suggest that the sites occupied by the substrates CDNB and mBBr are not identical.
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, the modification does not appreciably affect the apparent Km for either CDNB or glutathione; normal Michaelis-Menten kinetics was observed. For the 30-min sample, the apparent Km for mBBr was also determined yielding a value of 47 ± 8 µM, only slightly higher than that of native GST (33 ± 3 µM) cited previously.
In addition, we tested the ability of this 30-min mB-modified enzyme (with 13% residual activity toward mBBr) to bind the hydrophobic compound 8-anilino-1-naphthalene sulfonate (ANS; Fig. 1), using the fluorescence enhancement of enzyme-bound ANS. Figure 5 shows that the binding of ANS to GST is the same for both control GST (KD = 5.6 µM) and modified GST (KD = 5.4 µM). These results indicate that ANS occupies a hydrophobic binding site distinct from the mBBr site, and that covalent modification with mBBr does not affect the binding of ANS to GST .
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was incubated with mBBr for 30 min in the presence of either of two protectants (as in the samples of Table 2), and the kinetic properties of the isolated enzymes were determined with either CDNB or mBBr as substrate. When the glutathione site was protected with S-methylglutathione, the enzyme had 43% residual activity, and the apparent Km values for mBBr and CDNB were 43 ± 9 and 1020 ± 26 µM, respectively. Alternatively, when the xenobiotic substrate region was protected with S-(hydroxyethyl)bimane, the enzyme had 72% residual activity and exhibited apparent Km values for mBBr and CDNB of 44 ±8 and 910 ± 13 µM, respectively. These data show there is no appreciable change in Km for mBBr or CDNB in these "protected enzymes" suggesting that modification at one site inactivates that site, but does not influence the kinetic characteristics of other unmodified sites.
Isolation and characterization of peptides from mB-modified GST
GST (0.3 mg/mL) was inactivated for 30 min by 0.15 mM mBBr at pH 7.0 and 25°C. The resulting modified enzyme, with 13% residual activity toward monobromobimane, was isolated, incubated with urea and N-ethylmaleimide to block unreacted cysteine residues, dialyzed, and digested with trypsin. The digest was subjected to HPLC separation using a C18 column and 0.1% trifluoroacetic acid as a solvent system with an acetonitrile gradient, as illustrated by Figure 6A. Three peptide regions, designated I, II, and III, exhibit the characteristic bimane fluorescence (Fig. 6B).
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shows the results of peptide sequencing. Peak I contained two peptides, mB-C-K corresponding to residues 99–100, and HLGR corresponding to residues 69–72 in the known amino acid sequence of pig lung glutathione S-transferase . Cys99 in cycle 1 of the Edman degradation, was designated as the residue modified by mBBr because of its characteristic PTH peak, which appears between the PTH derivatives of Tyr and Pro as demonstrated by Hu and Colman using mB-Cys that they synthesized (Hu and Colman 1995; Hu et al. 1996). In contrast, when cysteine is modified by N-ethylmaleimide, the product can be observed as a distinct doublet appearing between PTH-Pro and PTH-Met as reported by Smyth and Colman (1991). Additional evidence for the chemical modification of peptide CK in peak I was provided by electrospray mass spectrometry. Peptides having masses of 482.88 amu (ca. 14% of total), 252.41 amu (ca. 68% of total), and 439.48 amu (ca. 10% of total) were detected. The mass of 482.88 amu is identical to the predicted mass of the contaminating tetrapeptide in peak I (His-69 to Arg-72), the mass of 252.41 amu is that of the unmodified form of the peptide (Cys-99 to Lys-100), while the mass of 439.48 amu corresponds to the predicted mass of mB-C-K in which the bromide group of mBBr had been displaced by the thiol group in Cys99. Although mB-C-K was not stable to electrospray mass spectrometry, sufficient adduct survived to directly demonstrate chemical modification of the peptide.
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. Both peptides had many potential amino acid targets for mBBr modification. Thus, this peptide peak was digested with V8 protease and was then subjected to the same HPLC solvent system as before (Fig. 6A). Fluorescent peak IIa eluted at 139 to 140 mL, earlier than Peak II. Peak IIa contains peptide TWPPLKPS-mB-C45LFR, corresponding to amino acids 37–48 of the known sequence. Assignment of Cys45 as the residue modified by mBBr was again made by the characteristic peak appearing between the PTH derivatives of Tyr and Pro (Hu and Colman 1995; Hu et al. 1996). Peak III contains peptide CEAMR, corresponding to residues 14–18 in the known amino acid sequence, with Cys14 modified. Assignment of modified residues was confirmed by the elution profile of PTH-mB-modified amino acid standards. Thus, in the absence of protectants, mBBr labels Cys99 (Peak I) and Cys45 (Peak II) as the major reaction products present in approximately equal amounts, with a minor amount of modification of Cys14 (Peak III).
Effect of protectants on fluorescent peptide peaks
Table 5, lines 1 to 3, records the effect on the magnitude of the three fluorescent peaks of increasing the incubation time of mBBr with the enzyme, in the absence of protecting ligands. At all times, approximately equal amounts of fluorescence are found in Peaks I (46–49%) and II (36–45%), with much less fluorescence in Peak III, although the total incorporation increases from 10 to 30 min. Table 5, lines 3 to 6, compare the amount of incorporation into these three peaks at a single time, 30 min, in the absence and presence of ligands. Compared to the absence of ligands (Table 5, line 3), a markedly lower incorporation was found in Peptide II when S-methylglutathione was present in the reaction mixture (line 4), indicating this peptide is within the glutathione binding region; a smaller percent decrease is seen in Peptide I. Comparison of lines 3 and 5 reveals the major decrease of mBBr incorporation is in Peptide I (although there is some effect on Peak II) when the enzyme is protected by S-(hydroxyethyl)bimane. This observation suggests that peptide I is primarily within the mBBr substrate binding site. When glutathionyl-bimane is added to the reaction mixture, there is a striking decrease in mBBr incorporation into both peptides I and II, suggesting the glutathione binding region and the mBBr site are not far apart. Throughout the different conditions investigated, peptide III was always modified, indicating that additional minor reaction occurs at an amino acid(s) not essential for enzymatic activity.
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are CDNB, ethacrynic acid, and stilbene oxide (Pettigrew et al. 1999, 2001). Here, we have shown that monobromobimane is also a substrate for GST . Overall, the enzyme exhibits higher affinity for mBBr, as reflected by the apparent Km, than for the often used xenobiotic substrate CDNB. The observation that S-(hydroxyethyl)bimane functions as a competitive inhibitor with respect to the substrate mBBr, but not the substrate CDNB, implies that the two substrate sites are distinct. Covalent reaction of GST with mBBr affects the enzyme’s ability to catalyze the reaction between glutathione and mBBr, with a lesser effect on its activity in catalyzing the conjugation of glutathione with CDNB. These results suggest that the sites occupied by the two substrates are close, but are distinguishable. Furthermore, after covalent reaction with mBBr, there is little or no effect on the affinity of the enzyme for CDNB.
The first crystal structures for GST showed that there are at least two binding sites per monomer (Sinning et al. 1993; Ji et al. 1997; Prade et al. 1997): The G-site is very specific for glutathione, whereas the binding site for the xenobiotic substrate (H-site) appeared less specific, in keeping with the ability of GSTs to react with a wide variety of toxic agents. In addition to their enzymatic roles, GSTs bind to large lipophilic molecules (>400 Daltons), leading to the proposal that GSTs are involved in the storage and rapid transport of these molecules in the aqueous phase of the cell (Litwack et al. 1971; Habig et al. 1974; Tipping and Ketterer 1981; Cacurri et al. 1990). These "ligandin" (nonsubstrate) sites were later shown to be a property of several GST isoforms including GST (Cacurri et al. 1990). Some of the ligands studied were sulfasalazine, cibacron blue, bromosulfophthalein (BSP), and 8-anilino-1-sulfonate (ANS; Bico et al. 1994; Oakley et al. 1999). "Ligandin" binding is characterized by noncompetitive inhibition towards CDNB (Ketley et al. 1975; Mannervik and Danielson 1988). Furthermore, the kinetic data suggested that the nonsubstrate or "ligandin" binding site(s) (the L-site) were distinct from the G- and H-sites. However, controversy exists as to whether ligandin sites of several GST isozymes are separate or the same as those occupied by the xenobiotic substrates (Lyon and Atkins 2002).
Monobromobimane, when added to the enzyme in the absence of glutathione functions as a specific, irreversible inactivator. Evidence for this statement is that mBBr inactivates the enzyme in a time-dependent manner, which is not reversed by gel filtration or dialysis. This reaction provides an opportunity to reevaluate the relationship among the various sites of glutathione S-transferase . The enzyme incorporates about 2 moles of monobromobimane/mole of subunit and is modified predominantly at Cys99 and Cys45. S-Methylglutathione decreases the reaction of mBBr with Cys45 suggesting this amino acid is at or near the glutathione binding site. The addition of S-(hydroxyethyl)bimane to the reaction mixture also partially protects the enzyme against inactivation and decreases incorporation into Cys99 implying this amino acid is at the mBBr site. Glutathionyl-bimane decreases the inactivation rate to zero and also decreases the mBBr reaction with both cysteines. These results show that the two sites of reaction are not far apart. Moreover, very minor incorporation was observed when both S-methylglutathione and S-(hydroxyethyl)bimane were in the reaction mixture. (Cys14 also reacted with mBBr but it is probably not at the catalytic site because protectants against inactivation do not decrease the incorporation levels in this residue.)
The dependence of the rate of inactivation on mBBr concentration was determined in the absence and presence of protectants to evaluate if binding of mBBr to GST occurs prior to modification, and to assess if modification of both cysteines is needed for complete inactivation. When either cysteine is protected, GST is still inactivated (albeit at lower rates) suggesting that it is not necessary to modify both cysteines to completely inactivate the enzyme (as measured by mBBr as substrate). In the presence of S-methylglutathione, kobs exhibits a hyperbolic dependence on the mBBr concentration, reaching a maximum rate constant for mBBr inactivation that is independent of the [mBBr]. These results suggest that mBBr binding is specific, and occurs prior to enzyme inactivation. The KI (127 µM) obtained from the kobs versus [mBBr] graph is somewhat higher than the Km (33 µM) for mBBr in the enzyme-catalyzed reaction with glutathione; this result may be attributable to the difference in the kinetic meaning between Km and KI, or it may indicate that mBBr has a slightly weaker affinity for the enzyme under the conditions of inactivation, perhaps because the enzyme at this site can bind mBBr in alternative orientations. In the presence of S-(hydroxyethyl)bimane, kobs exhibits a linear dependence on the mBBr concentration, suggesting that modification under these conditions occurs by a bimolecular chemical reaction, rather than by affinity labeling. In the absence of ligands, the kobs represents the sum of all the monobromobimane reactions, and the linear dependence of kobs on [mBBr] indicates that the bimolecular chemical reaction predominates.
These experimental results can best be understood in terms of the crystal structures of GST . In the human GST (hGST )1 crystal structure, represented in Figure 7A, glutathione is positioned in an orientation suitable for reaction with mBBr docked at the active site. The distance between the —CH2Br of mBBr and —SH of glutathione is approximately 1.78 Å, whereas it is about 8.97 Å to the —SH of Cys101. This must be the xenobiotic substrate site for monobromobimane. An alternate mode of docking of mBBr is pictured in Figure 7B, where Cys101 (equivalent to Cys99 in pig lung GST ) is positioned in a conformation suitable for reaction with mBBr. The distance between —CH2Br of mBBr and —SH of Cys101 is approximately 2.05 Å, whereas it is about 10.3 Å to the —SH of glutathione. This orientation explains the accessibility of mBBr to Cys101 and the different values obtained for monobromobimane’s Km and KI (from the mBBr concentration dependence of kobs). In solution, it is likely that mBBr can adopt either of these two conformations in the active site, and that is the reason both reactions are observed; that is, that mBBr is both a substrate and an irreversible inactivator of GST . There are ample precedents in the literature for a small molecule binding to a enzyme in two different orientations. Most relevant is the report of two modes of binding of the inhibitor ethacrynic acid to GST , based on analysis of crystal structures of complexes of GST (Oakley et al. 1998).
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To evaluate whether mBBr is really in a different location from the dinitrophenyl group, the structure of hGST P1-1 complexed with dinitrobenzyl-glutathione was overlaid on the structure of the enzyme with monobromobimane docked into the substrate site as in Figure 7A. The overlay is shown in Figure 7D. It is clear that mBBr and dinitrophenyl are in two discrete locations, indicating that the site for mBBr, when docked in this orientation, is distinct from the CDNB site.
We have demonstrated that monobromobimane reacts covalently with a second cysteine. In the model shown in Figure 7C, we have docked mBBr into a site close to Cys47 (equivalent to Cys45 in pig lung GST ). The distance between —CH2Br of mBBr and —SH of Cys47 is approximately 1.80 Å. This short distance can account for the reaction of mBBr at this residue. Moreover, this residue is only about 4 Å away from the glutathione molecule, thereby explaining the protection provided by S-methylglutathione against reaction of mBBr with Cys47. Furthermore, in a previous study conducted by Park et al. (2001), substitution of Cys47 by Ser decreased the binding affinity fivefold between hGST and glutathione. Also, Nishihira et al. (1992) found that reaction of Cys47 with the bulky reagent 7-fluoro-4-sulfamoyl-2,1,3-benzodiazole inactivates hGST by steric hindrance of the glutathione binding site. In addition, Caccuri et al. (1992) reported that the human GST is inactivated by 1-chloro-2,4-dinitrobenzene upon modification of Cys47, and that protection is provided by S-methylglutathione. These results are all consistent with the location of Cys47 at or near the glutathione binding site.
To address the question of whether mBBr reacted within a "ligandin" site, we evaluated 8-anilino-1-naphthalene sulfonate binding by native GST and by enzyme that had been covalently modified by mBBr. The results revealed that ANS bound equally well to the mBBr-modified and unmodified enzyme, indicating that the hydrophobic "ligandin" site is distinguishable from the xenobiotic substrate binding site. Furthermore, we considered the possibility that mBBr was occupying the site of another large hydrophobic, nonsubstrate molecule, bromosulfophthalein (BSP), which is a noncompetitive inhibitor with respect to CDNB (Bico et al. 1994). Thus, the mBBr was docked in the bromosulfophthalein site in hGST (not shown). The distances between the —CH2Br of mBBr at the BSP site and —SH of Cys101, —SH of Cys47, and glutathione are 16.6 Å, 12.9 Å and 8.1 Å, respectively. These long distances indicate that mBBr cannot be located at the BSP position and react with either Cys101, Cys47 or enzyme-bound glutathione.
We reported previously that mBBr acts as an affinity label of the 3-3 (mu-class) and 1-1 (alpha class) glutathione S-transferases. For the µ class enzyme, Tyr115 is the target residue whose modification correlates with loss of enzymatic activity towards CDNB. Inactivation of the -class 1-1 isozyme occurs by modification of Cys17 and Cys111, residues found within the steroid binding site of this isozyme. The amino acids modified by mBBr in the µ and class GSTs are not homologous to those targeted in the GST. These results demonstrate that the three classes of glutathione S-transferase (, µ, and ) react in a distinctive manner with monobromobimane. Differences in the binding site structures are responsible for the characteristic specificities of these three glutathione S-transferase isozymes.
In summary, the results presented in this paper indicate that GST has at least two different xenobiotic substrate sites—one for mBBr, and one for CDNB. Monobromobimane, in addition to serving as a substrate for GST , acts as an affinity label. Modification of Cys99 occurs from the xenobiotic substrate site occupied by mBBr during its reaction with glutathione, while reaction with Cys45 takes place with monobromobimane positioned at or near the glutathione binding site.
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Enzyme purification
GST was purified from pig lung as described previously (Pettigrew et al. 1999) and was stored (in 0.1 M potassium phosphate buffer, pH 6.5) frozen at -80°C, conditions under which the enzyme is stable. The procedure yields GST at greater than 90% purity as judged by reverse-phase HPLC (Vydac C4 reverse-phase column, equilibrated with 50% acetonitrile containing 0.075% TFA and using a 50-min gradient of 50–100% acetonitrile containing 0.075% TFA).
Enzyme assays
Enzymatic activity toward 1-chloro-2,4-dinitrobenzene was measured using a Hewlett-Packard 8453 spectrophotometer by monitoring the formation of the conjugate of CDNB (1 mM) and glutathione (2.5 mM) at 340 nm (
= 9.6 mM-1 cm-1) in 0.1 M potassium phosphate buffer, pH 6.5, at 25°C according to the method of Habig et al. (1974). All measurements were corrected for the spontaneous nonenzymatic rate of formation of the conjugate of glutathione and CDNB.
For mBBr, the enzymatic activity was measured using a Perkin-Elmer MPF-3 fluorescence spectrophotometer (excitation at 390 nm and emission at 480 nm) by monitoring the formation of the conjugate of mBBr (100 µM) and glutathione (600 µM) in 0.1 M potassium phosphate buffer, pH 6.5, at 25°C according to the method of Hulbert and Yakubu (1983). A known amount of glutathione-bimane was used as a fluorescence standard to calibrate and calculate the product formation. We thank Jennifer L. Hearne for the gift of glutathione-bimane that she synthesized.
To determine the apparent Km value of glutathione, a range of glutathione concentrations (0.01 to 2 mM) was investigated at a constant CDNB concentration (3 mM). Similarly, the apparent Km for CDNB was determined from a range of concentrations of CDNB (0.01 to 3 mM) at a constant glutathione concentration (2.5 mM) in 0.1 M potassium phosphate buffer, pH 6.5. For the determination of the Km of mBBr, the glutathione concentration was maintained at 600 µM while mBBr was varied from 10 to 200 µM. Data were analyzed by fitting directly to the Michaelis-Menten equation using a nonlinear curve-fitting program (SigmaPlot from SPSS), and Km values are reported along with their standard errors.
Reaction of GST with mBBr
GST (0.3 mg/mL) was incubated in 61 mM phosphate buffer, pH of 7.0 at 25°C with various concentrations of mBBr by the addition of appropriate stock solutions of mBBr in DMF. The volume of DMF was maintained at 10% of the total volume of the reaction mixture. In control experiments, enzyme was incubated under the same conditions including 10% DMF but without mBBr. In every case, aliquots of the reaction mixture were removed at specified times and assayed for enzymatic activity using either CDNB or mBBr as substrates. For the CDNB assay, aliquots of the reaction mixture were diluted 15-fold with 0.1 M potassium phosphate buffer, pH 6.5 at 25°C, and assayed by the addition of 20 µL to the cuvette. For the mBBr assay, aliquots of the reaction mixture (15 µL) were assayed without diluting. The rate constants for reaction of the enzyme with mBBr were calculated from semilogarithmic plots of E/E0 versus time, in accordance with the pseudo first-order kinetic equation:
 |
where E0 is the activity of the enzyme at time zero, Et represents the activity at a given time, t, and kobs is the observed pseudo first-order rate constant.
In the preparation of modified and control enzyme, excess unreacted reagent was removed from the reaction mixture by the gel filtration procedure of Penefsky (1979). Aliquots (0.5 mL) of the reaction mixture at a given time were applied to a 5-mL column of Sephadex G-25 equilibrated with 0.1 M potassium phosphate buffer, pH 7.0. The protein concentration in the filtrate was determined by the Bio-Rad protein assay, which is based on the dye-binding method of Bradford (1976), using a Bio-Rad 2550 RIA reader (600-nm filter). Purified GST was used to establish the standard protein concentration curve for these determinations.
Measurement of incorporation of mBBr into GST
GST (0.3 mg/mL) was incubated for the indicated time with 0.15 mM mBBr, with or without the addition of protectants under standard reaction conditions. Excess reagents were removed by gel filtration columns, and the protein concentration was determined by the Bio-Rad method, as described above. The amount of reagent incorporated was determined from the absorbance at 390 nm using 390nm = 5360 M-1 cm-1, which is the characteristic absorbance for the bimane moiety in model compounds such as mB-SG (Kosower and Kosower 1987).
Preparation of modified GST for fluorescence and kinetic studies
GST (0.3 mg/mL) was incubated for 30 min with 0.15 mM mBBr under standard reaction conditions. Excess reagents were removed by gel filtration and the protein concentration was determined by the Bio-Rad dye-binding method. The prepared modified enzyme was frozen quickly and stored at -80°C.
Measurement of ANS-binding to mB-modified GST
Equilibrium-binding studies with ANS were determined by measuring the enhancement of ANS fluorescence when the ligand (0–45 µM) binds to native or mB-modified GST (16 µM), as described by Bico et al. (1994). Excitation was at 390 nm and emission at 480 nm. Fluorescence measurements were made in 0.1 M phosphate buffer, pH 6.5 at 25°C.
Trypsin digestion of mB-modified GST
GST (0.3 mg/mL) was incubated for the indicated time with 0.15 mM mBBr with or without the addition of ligands under standard reaction conditions. Excess reagent was removed by gel filtration as described above. Solid urea (to give 6 M as the final concentration) and N-ethylmaleimide (10 mM) were added to the enzyme and incubated for 30 min at 25°C. The enzyme solution was then dialyzed overnight against 50 mM ammonium bicarbonate, pH 7.8 (4 L).
The enzyme solution was lyophilized and then resolubilized in 250 µL of 8 M urea in 50 mM ammonium bicarbonate, pH 7.8. This solution was incubated at 37°C for 2 h to denature the protein. Ammonium bicarbonate (750 µL, 50 mM, pH 7.8) was then added to the solution to dilute the urea to 2 M. Trypsin [2.5% (w/w)] was added and the enzyme sample was incubated for 1 h at 37°C. A second aliquot of the trypsin solution was added and incubation was continued for another 1 h at 37°C.
HPLC separation of modified peptides
The tryptic digest was injected onto a Varian 5000 LC HPLC (Varian) equipped with a Vydac C18 reverse-phase column (0.46 x 25 cm). At a flow rate of 1 mL/min, the peptides were separated by a 225-min linear gradient from 0 to 45% solvent B (solvent A, 0.1% TFA in water; solvent B, 0.075% TFA in acetonitrile), followed by a 30-min gradient to 100% solvent B. The eluate was monitored at 220 nm with 1-mL fractions collected. Aliquots of each fraction were tested for fluorescence (excitation at 390 nm and emission at 480 nm).
Peak II from the trypsin digest was pooled, lyophilized, and redissolved in 900 µL of 0.1 M phosphate buffer, pH 7.8. Staphylococcus aureas V8 protease was added once at 0.25% w/w and the solution was incubated at 25°C for 2 h. The redigested sample was applied to the HPLC (Vydac C18 column) and eluted at a flow rate of 1 mL/min using the same gradient as before.
Analysis of isolated peptides
The amino acid sequences of the purified peptides were determined using an Applied Biosystems Procise protein/peptide sequencer. Cysteine modified by N-ethylmaleimide (S-[N-ethylsuccinimido]cysteine) was identified by a doublet migrating on the HPLC column of the sequencer between the PTH derivatives of Pro and Met (Smyth and Colman 1991) and mB-Cys by a distinct peak appearing between the PTH derivatives of Tyr and Pro. The amount of mB-Cys in picomoles was estimated using the PTH derivative of Pro, as standard.
Electrospray mass spectrometry
Peak I from the HPLC separation (as described previously) was lyophilized and redissolved in 0.1% TFA/H2O. Peptide molecular weights were determined using a Micromass Autospec Q mass spectrometer equipped with an electrospray attachment.
Molecular modeling
Modeling was conducted using the program Insight II from Biosym Technologies on a Silicon Graphics workstation. The molecular model of mBBr was built and energy minimized using the Builder module of the Insight II program. The mBBr molecule was positioned into a site close to Cys101 of human GST (PDB nos. 19GS and 18GS) by sequentially rotating and translating it along the x, y, and z axes, and the intermolecular energy in terms of both van der Waals’ and electrostatic interactions, as well as the interatomic distance between the sulfur atom of Cys101 (equivalent to Cys99 in pig lung GST ) and the bromine-bearing carbon atom of mBBr were continuously monitored for conformations with reasonable distances and potential energies constituting possible productive interactions for chemical modification of the cysteinyl thiol group. Another possible conformation for the molecule was constructed by rotating mBBr so that the bromine-bearing carbon atom would face the thiol group of glutathione. The mBBr was also docked at a site close to Cys47 of human GST (equivalent to Cys45 in pig lung GST ). One other model was constructed by docking mBBr at the bromosulfophthalein site. The manually docked complex, as well as mBBr modified enzyme models were submitted to the DiscoverTM program from Biosym for extensive energy minimization using steepest descent and conjugate gradient methods to relieve residual van der Waals’ overlaps and optimize the structures.
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