Cross-Linking of Human Placenta π Class Glutathione S-Transferase

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Chem. Res. Toxicol. 1996, 9, 1044-1049

Cross-Linking of Human Placenta π Class Glutathione S-Transferase Dimer by Chlorambucil Yetrib Hathout, Torria Ellis, Daniele Fabris, and Catherine Fenselau* Structural Biochemistry Center, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 5401 Wilkens Avenue, Baltimore, Maryland 21228 Received November 20, 1995X

Interaction of chlorambucil and the glutathione-depleted human placenta π class glutathione S-transferase (πGST) results in the formation of a complex between the drug and the protein at physiological pH. This complex is not formed in the presence of glutathione or Shexylglutathione. Molecular mass measurement of the reaction product using matrix-assisted laser desorption mass spectrometry indicates that one molecule of chlorambucil cross-links two subunits of the homodimeric protein. A combination of enzymic proteolysis, high performance liquid chromatography, and mass spectrometry reveals that chlorambucil alkylation occurs at cysteine 47 of one subunit and cysteine 101 of the second subunit. This result supports the idea that conformational changes occur in glutathione-depleted πGST, which allow the bifunctional tether of chlorambucil to cross-link the two subunits of the protein.

Introduction (GSTs)1

Glutathione S-transferases are a family of ubiquitous enzymes that play an important role in cellular detoxification. These enzymes catalyze the conjugation of reduced glutathione (GSH) with a large variety of xenobiotic agents (1, 2), including the anticancer drug chlorambucil (3). Mammalian GSTs have been classified into five major groups, named R, µ, π, θ, and microsomal GST, based on their primary structure and immunogenicity and substrate and inhibitor specificity (2). Crystal structures have been determined for the first three enzymes in complexes with GSH analogues, which show that these isoenzymes comprise homo- or heterodimers formed by noncovalent association of two subunits (4). Each subunit contains one site for the cofactor GSH (G-site) and one site for the substrate (Hsite). A detailed description of the structure of human placenta πGST complexed with S-hexylglutathione has been provided (5). This protein is formed by the association of two identical subunits, each subunit consisting of 209 amino acid residues. Each subunit contains four reduced cysteine residues at positions 14, 47, 101, and 169. One characteristic of this GST class is that Cys-47 and Cys-101 in each subunit are proximal to the cofactor and the substrate binding sites. Chemical modifications of Cys-47, a highly conserved residue in πGSTs from different sources, by thiol group modifiers (6) or by oxidation (7, 8) are reported to inhibit the catalytic activity of πGST. Kinetic studies have demonstrated that either the cofactor GSH or the substrate may be bound first by glutathione S-transferases (4, 9, 10) and that πGST undergoes localized structural or conformational changes when GSH is and is not bound. These include kinetic studies of nonsubstrate lipid binding (11) and of GSH Abstract published in Advance ACS Abstracts, August 15, 1996. Abbreviations: GST, glutathione S-transferase; GSH, glutathione; CLB, chlorambucil; DTT, dithiothreitol; TFA, trifluoroacetic acid; MALDI; matrix-assisted laser desorption; HABA, 2-(4′-hydroxyazobenzene)benzoic acid; CHCA, R-cyano-4-hydroxycinnamic acid; FAB-MS, fast atom bombardment mass spectrometry; CNBr, cyanogen bromide; CID, collisionally-induced dissociation. X 1

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binding (2, 4, 12), which is proposed to operate by an induced fit mechanism (12). Limited proteolytic study further supports the occurrence of πGST in different conformations depending on whether GSH is bound or not (13). Unfortunately, crystal structures of πGST uncomplexed or complexed only with substrate have not yet been obtained that are suitable for X-ray crystallography. In the present work the conformation of πGST complexed with a substrate but not with GSH has been studied using an affinity labeling strategy. The chemotherapeutic mustards melphalan and chlorambucil have been found to function as affinity labels in characterizing specific binding sites on metallothionein (14, 15). Chlorambucil has been shown to be a substrate for πGST, apparently binding at the H-site (3), and is used in this study as affinity labeling agent to characterize the conformation of πGST free of GSH.

Experimental Section Caution: Cyanogen bromide and chlorambucil are carcinogenic products. Human placenta πGST purified by Sigma may contain infectious agents. These products should be handled carefully. Protein Preparation and Incubation Procedures. To obtain the fully active enzyme, human placenta πGST (Sigma Chemical Co., St. Louis, MO) was treated with dithiothreitol (DTT) at pH 8 in 0.1 M Tris-HCl and 1 mM EDTA for 60 min at 37 °C under argon. (DTT mol/GST mol ) 50/1) (7). The excess of DTT and unbound GSH was then removed by passing the solution through an Econo-Pac 10 DG column (Bio-Rad, Richmond, CA) equilibrated and eluted with degassed 0.1 M potassium phosphate buffer (pH 7). The recovered GST was shown to be active (90 µmol‚min-1‚mg-1) by assay with 1-chloro2,4-dinitrobenzene (1). The inactivation of CLB-alkylated πGST was determined by the same assay. Protein solution (43 µM in 0.1 M potassium phosphate buffer, pH 7) was preincubated at 37 °C under argon for 5 min. Then a solution of chlorambucil (CLB) in ethanol was added (CLB mol/GST mol ) 4/1 and ethanol 2% v/v). Incubation was continued for periods between 30 and 180 min. All experiments and control reactions reported were carried out with these conditions. Higher ratios of CLB/ GST were also used in two cases, as indicated. As a control

© 1996 American Chemical Society

Affinity Labeling of Apo-πGST reaction for each incubation, 43 µM of reduced GST was incubated with 2% ethanol under the same conditions. In addition, πGST was preincubated under denaturing conditions (6 M guanidinium chloride in 0.1 M potassium phosphate buffer, pH 7) and then incubated with CLB. To test whether or not GSH and its derivatives can affect the reaction of CLB with πGST, 1 mL aliquots of GST solution (43 µM in 0.1 M potassium phosphate buffer, pH 7) were incubated with 1 mM S-hexylglutathione and with 5 mM GSH for 15 min at 37 °C. Then CLB in ethanol was added (CLB mol/GST mol ) 4/1 and ethanol 2% v/v), and the incubation was monitored up to 3 h by HPLC. Similar conditions were used to optimize the formation of monoalkylated product, except that the molar ratio of CLB/GST was 20:1 and the reaction was terminated after 30 min. Aliquots from the GST/CLB reaction mixtures were analyzed by high performance liquid chromatography (HPLC) (LC-600 pumps, Shimadzu, Tokyo, Japan). CLB-modified GST and unreacted GST were separated on a reverse phase C18 column (Aquapore RP-300 7 µm, 250 × 4.6 mm, Applied Biosystems, San Jose, CA). UV detection was carried out at 215 nm, and the gradient solvent was 0.08% trifluoroacetic acid (TFA) in acetonitrile developed from 30% to 60% in 55 min in 0.1% TFA in water. The collected peaks were either freeze-dried or directly subjected to mass spectrometric analysis. In a duplicate experiment, production of the glutathione conjugates of CLB was monitored by HPLC using the same column as above. In this case, the eluent was 0.1 M ammonium acetate in 5% methanol and the gradient was 0.1 M ammonium acetate in 90% methanol developed from 0% to 100% in 40 min. The products were detected by UV absorbance at 254 nm. They were identified by comparison of their retention times to those of the products characterized from the chemical reaction of reduced glutathione (1 mM) with CLB (500 µM) in 0.1 M potassium phosphate buffer, pH 7 (16). Identification of the Chlorambucil Alkylation Sites in GST. CLB-treated GST (1 mg) and untreated GST (1 mg) were dissolved in a denaturing buffer (0.1 M Tris-HCl, pH 8, 1 mM EDTA, 6 M guanidinium chloride), and the free cysteines were alkylated with iodoacetamide (50 molar equiv) in the dark for 1 h at 37 °C under argon. The excess of reagent was removed by gel filtration using an Econo-Pac 10DG column equilibrated and eluted with 20 mM ammonium bicarbonate (pH 8). The recovered proteins were digested with TPCK-treated trypsin (Sigma Chemical Co., St. Louis, MO) for 4 h at 37 °C under argon or with CNBr (17) in 200 µL of 70% TFA for 24 h at room temperature. The resulting peptide mixtures were purified by HPLC using the Aquapore C18 column, and solvent gradient 0.08% TFA in acetonitrile was developed from 10% to 60% in 45 min in 0.1% TFA in water. The collected peptides were freeze-dried and stored at -20 °C for mass spectrometry analysis. Mass Spectrometry. Matrix-assisted laser desorption mass spectra were obtained using a Kompact MALDI III (Kratos, Manchester, U.K.) reflectron time-of-flight mass spectrometer, with a 337 nm nitrogen laser and 20 kV acceleration voltage. For πGST, desorption was achieved using 2-(4′-hydroxyazobenzene)benzoic acid (HABA) as matrix with an analyte/matrix ratio of 1/5000. Protein, 0.3 µL of 10-20 µM solution, was mixed with 0.3 µL of 50 mM matrix solution in 70% acetonitrile and 30% (0.1% TFA/water). Bovine R-chymotrypsinogen was used as external or internal calibrant. For HPLC purified tryptic peptides, R-cyano-4-hydroxycinnamic acid (CHCA) was selected as the matrix with an analyte/matrix ratio of 1/10 000. Prepronerve growth factor (2003.3 Da) and protonated matrix dimer were used as internal or external calibrants when the instrument was operated in linear or reflectron mode, respectively. Fast atom bombardment mass spectrometry (FAB-MS) was performed on a HX110/HX110 high resolution four-sector mass spectrometer (JEOL, Tokyo, Japan) with a FAB gun operated at 6 kV. The matrix was an equimolar mixture of glycerol and monothioglycerol. Samples were dissolved first in 0.1% TFA in water, and then 1 µL of solution was mixed with the matrix on the probe tip. High energy collision-induced dissociation

Chem. Res. Toxicol., Vol. 9, No. 6, 1996 1045

Figure 1. Reversed phase HPLC analysis of products from reaction of CLB with human placenta GSTπ. (a) GST was incubated with ethanol alone in 0.1 M potassium phosphate buffer, pH 7 for 2 h; (b) GST was incubated with ethanol containing CLB in the same buffer for 2 h. (CID) of selected singly-charged precursors was accomplished in the third field free region inside a 4 kV floated collision cell. Helium was used as the collision gas at a pressure sufficient to attenuate the ion beam by 60%. Electrospray ionization mass spectrometry was performed with an Analytica of Branford ion source (Branford, CT) fitted to JEOL (Tokyo, Japan) HX110/HX110 four-sector mass spectrometer. Analytes were dissolved in a solution of water, methanol, and glacial acetic acid in 49:49:2 ratio and injected into the electrospray source by a Harvard (South Natick, MA) syringe pump at a flow of 1 µL/min.

Results The HPLC analysis of πGST from the 120 min control reaction is shown in Figure 1a. The protein is eluted as a single peak and remains unchanged after 3 h incubation in buffered medium at 37 °C, whereas protein recovered from the reaction of CLB with πGST was resolved into two peaks, one major peak 1 with a retention time similar to that of GST from the control reaction and a smaller peak 2 with a longer retention time (Figure 1b). The transformation of πGST by CLB was monitored from increases in the area of peak 2. As shown in Figure 2 (solid line) modification of the enzyme proceeded slowly during the first 30 min of incubation. Then a significant increase was observed between 30 and 60 min, followed by a slow reaction after 60 min. When a fresh solution of CLB was added to the reaction mixture after 2 h (data not shown), additional protein was found to be transformed. Presumably this reflects loss of CLB in a competing reaction with water in the buffered medium at 37 °C (18). Conjugation of CLB with glutathione was also observed, although no GSH was added to the incubation. Two conjugated products were identified by comparison of their retention times with those of products formed in a parallel chemical reaction of GSH

1046 Chem. Res. Toxicol., Vol. 9, No. 6, 1996

Figure 2. Time course for conjugation of CLB with glutathione (dashed line) and alkylation of πGST (solid line) in the incubation of 172 µM CLB and 43 µM πGST. No glutathione was added.

with CLB. The chemical products were characterized by FAB mass spectrometry (16) as the monoglutathionyl adduct of CLB and the monohydroxy monoglutathionyl product. The combined formation of these two GSH conjugates in the GST incubation increased dramatically between 0 and 15 min and then leveled off (Figure 2, dashed line). This indicates that residual GSH was still bound to πGST purified by gel filtration and that GSH had to be depleted before protein modification commenced. The concentration of GSH in the πGST solution was estimated to be around 34 µM calculated on the basis of the percentage of CLB-GSH conjugates formed from the total CLB (172 µM) added to 43 µM πGST solution. Modification of πGST is detected only after GSH is depleted (Figure 2, solid line). The modification of the protein could not be detected when the enzyme was preincubated with 5 mM GSH or 1 mM S-hexylglutathione, even when the ratio of CLB/ GST was increased to 10/1. These results suggest that the CLB target site in πGST is protected in the presence of GSH or S-hexylglutathione. Conversely, the specific activity of the enzyme dropped from 90 to 65 µmol‚ min-1‚mg-1 during a 120 min incubation with CLB. This loss of 28% of the activity corresponds well to the alkylation of approximately 23% of the protein (Figure 2) and indicates that alkylation inactivates catalytic activity. Molecular Mass Measurement. The HPLC separated protein products from the 120 min CLB-πGST reaction were analyzed by MALDI-TOF MS. 2-(4′Hydroxyazobenzene)benzoic acid was found to be an effective matrix for the desorption of this class of protein. Good mass accuracy (0.05-0.1%) was obtained when R-chymotrypsinogen was used as the internal calibrant. Figure 3a shows the MALDI spectrum of HPLC fraction 1 (recorded without the internal calibrant for clarity). The observed molecular mass of 23 236 ( 9 Da (average calculated from 6 different runs) corresponds to that of the unreacted πGST monomer and agrees with the calculated molecular mass (23 225 Da) from the protein

Hathout et al.

Figure 3. Matrix-assisted laser desorption mass spectra of the HPLC purified CLB-GST reaction product (Figure 1b). (a) Peak 1; (b) peak 2. Matrix used HABA.

sequence (19). Electrospray ionization mass spectrometry provided a consistent mass determination, 23 222 ( 3 Da. A MALDI mass spectrum of HPLC fraction 2 is shown in Figure 3b, in which a series of peaks related to πGST protein are observed. The peak at m/z 46 686, is very intense, and its mass is about 230 Da higher than the observed molecular mass of the protonated dimer complex in Figure 3a. This is consistent with addition of one molecule of CLB to two subunits of πGST and loss of two molecules of HCl. In addition, a new peak is detected at m/z 15 558, representing the triply charged ion corresponding to a molecular mass of 46 685 Da for the neutral alkylation product. The peaks detected at m/z 23 372 and 11 672 are assigned, respectively, as doubly- and quadruply-charged ions of molecular mass of 46 685 Da. An aliquot of HPLC fraction 2 was treated with DTT for 1 h at 37 °C, pH 8. Its MALDI mass spectrum was similar to that obtained before treatment, excluding the possibility that the peak at m/z 46 686 revealed formation of intersubunit disulfide bonds in πGST protein during incubation. In an additional experiment, starting material and products of the CLB-GST reaction were analyzed by SDS-PAGE. Under denaturing conditions, GST from the control reaction showed a single band at approximately 27 kDa, while the incubation mixture showed bands around 27 and 48 kDa. These results indicate that the CLB molecule covalently bonds to the protein by joining its two subunits. In another control reaction, mass measurement of protein recovered from the reaction of CLB with denatured πGST revealed formation of mono-, di-, and trialkylated mixture products with molecular masses of 23 484 ( 3, 23 749 ( 5, and 24 023 ( 5 Da, respectively. No cross-linked product was detected in this case. Characterization of the CLB Alkylation Sites in πGST. To identify the CLB target sites in cross-linked πGST, a mixture of CLB-alkylated and unalkylated

Affinity Labeling of Apo-πGST

Figure 4. Reversed phase HPLC analysis of peptides produced by trypsin from GST control (a) and from the CLB treated GST. (In the reaction of CLB with GST both reacted and unreacted GST are present and were digested by trypsin.) A peptide whose abundance is reduced in the digest of CLB treated GST is indicated by X; a new peptide in the digest of CLB treated GST is indicated by Y.

protein was recovered from the incubation medium by gel filtration. The remaining free cysteine thiolate groups were blocked by reaction with iodoacetamide, and the protein mixture was digested with trypsin. The same procedure was carried out with protein from the GST control reaction. HPLC profiles of tryptic peptides resulting from proteolysis of the GST control and the CLB-GST reaction are shown in Figure 4a and 4b, respectively. The chromatograms are similar, with the exception that peptide X decreased in CLB treated GST while a new peptide Y appeared (Figure 4b). The MALDI mass spectra of these two purified peptides are shown in Figure 5. Peptide X has a molecular mass of 1135.3 Da, corresponding to the tryptic fragment [45-54], while peptide Y has molecular mass of 1559.7 Da, interpreted as a modified product of peptide [45-54] . Precedent shows that nitrogen mustards react readily with cysteine (14, 15), and Cys-47 was tentatively assigned as the target site in peptide [45-54]. Because CLB cross-links the two GST subunits, cysteine-containing tryptic peptides from the other subunit were evaluated to provide a cross-linked peptide with the observed molecular mass (1560.7 Da). The fragment [101-102], which contains Cys-101, provides the requisite mass. The structure of this CLB cross-linked peptide is shown in the inset in Figure 6. Tryptic peptides were mapped by MALDI-TOF MS to account for greater than 90% of the sequence (19) in both πGST and the drug-alkylated product. No other peptides from the drug-alkylated protein were found to be modified in five separate experiments. To examine the structure of the CLB-modified peptide in more detail, an aliquot of the collected fraction was

Chem. Res. Toxicol., Vol. 9, No. 6, 1996 1047

Figure 5. Matrix-assisted laser desorption mass spectra of the HPLC purified peptide from the tryptic proteolysis of the CLBGST reaction product mixture. (a) Nonmodified peptide where cysteine was carboxamidomethylated; (b) CLB-modified peptide. Protonated peptide PNGF and the protonated matrix dimer (Rcyano-4-hydroxycinnamic acid) were used to calibrate each spectrum.

Figure 6. Mass spectrum of the collisionally-induced dissociation product ions from the CLB-modified peptide.

esterified with methanol (20) and analyzed by MALDI. The molecular mass of the this peptide shifts from 1559.7 to 1601.2 Da in agreement with the methylation of three carboxylic groups, two from the C-termini of the two peptide chains and one from the CLB moiety. For further confirmation, 4 mg of πGST was reacted with CLB under the conditions described above and a larger amount of CLB-modified peptide was collected for analysis by tandem mass spectrometry. The CID mass spectrum of the CLB-modified peptide is shown in Figure 6. The fragmentation pattern indicated on the figure for the mustard moiety (1078.8, 1105.8, 1310.8, 1342.9) has

1048 Chem. Res. Toxicol., Vol. 9, No. 6, 1996

Hathout et al.

Table 1. Molecular Masses of Peptides Generated by CNBr from πGST Treated with CLB for 30 min, and from Untreated πGST GST control (Da)

CLB-treated GST (Da)

assignmenta

calcd MW (Da)

2204 ( 1 2194 ( 1 8095 ( 4

2205 ( 0.5 2195 ( 0.4 8094 ( 3 8304 ( 2 13093 ( 2

[1-19]b [1-19]c [20-91]b [20-91]d [92-209]

2206 2196 8095 8306 13092

13092 ( 2

a All peptides were detected as homoserine lactone, except for the C-terminal peptide [92-209]. b Cysteines residues are carboxamidomethylated. c Cysteine residue transformed to cysteic acid during the experiment. d Cysteine residue alkylated with CLB.

been previously reported (14, 15) to account for the most abundant ions in the spectra of peptides modified by nitrogen mustards. Fragment ions formed by cleavage of the peptide backbone have been observed to be less abundant (14, 15), as is also seen in Figure 6. Backbone fragment ions are labeled a, d, x, y, v, and w, following the convention of Biemann (21). Formation of these sequence series ions is illustrated in the inset structure. The peaks designated P, L, and K at m/z values below 150 correspond to immonium ions formed from some of the amino acids (proline, leucine, and lysine) in the peptide. The fragment ions detected are consistent with assignment of cysteine as the alkylation site in the longer peptide. Ions in the w series are formed by cleavage in the amide backbone and by loss of the side chain from the newly released residue. Thus w8 ions are formed by loss from the precursor peptide of Ala-Ser and the alkylated side chain of Cys. The w7 ions are formed by loss of Ala-Ser-Cys and the side chain of Leu. The alkylation site in the shorter peptide is assigned as Cys101 because trypsin recognized unaltered Lys-102 for cleavage. After incubation for 120 min, only one product, the cross-linked dimer, was detected by molecular mass and among the tryptic peptides. The selectivity of the crosslinking or double alkylation sites is consistent with the expectation that CLB would bind as a substrate and then alkylate reactive sites in the protein that are accessible, i.e., an affinity labeling experiment. The great difference in the alkylation products detected from the reaction of CLB with denatured πGST under otherwise analogous conditions also supports the conclusion that both alkylation reactions took place in the dimer with the conformation associated with substrate binding in the absence of cofactor. A final experiment was carried out to characterize the initial site(s) of the first alkylation step in the crosslinking sequence. This experiment was terminated after 30 min (see Experimental Section) and analyzed by HPLC. Product formation was low; however, the major product was found by MALDI TOF to have a molecular mass of 23 484 ( 3 Da. This molecular mass corresponds to a monoadduct formed with elimination of one molecule of HCl. The product mixture was carboxamidomethylated and cleaved by CNBr. Peptide products were purified by HPLC and their molecular masses measured by ESI-MS. The results are summarized in Table 1. Among the three CNBr generated fragments from the CLB treated πGST, only peptide [20-91] was found to be chemically modified. The observed molecular mass of 8304 ( 2 Da is 266 Da higher than the molecular mass of 8038 Da for the unmodified peptide [20-91]. The

difference in mass corresponds to linkage with CLB with the loss of one HCl. This polypeptide contains only one cysteine, and that residue at position 47 was assigned as the site of initial alkylation. No other modifications were detected.

Discussion Chlorambucil, a chemotherapeutic agent with two reactive chloroethyl groups cross-links and inactivates the human placenta πGST monomers by alkylating Cys47 of one monomer and Cys-101 of the second monomer. This reaction occurs only in GSH-depleted πGST, and it is prevented in the presence of S-hexylglutathione. Derivatization of πGSTs by thiol group modifiers has been extensively studied by others (6, 22-24). It has been demonstrated that alkylation often occurs at Cys47 (human πGST) or Cys-45 (porcine πGST). Such a reaction causes a loss of enzyme activity, presumably by blocking or disturbing the conformation for GSH binding. Similarly, binding of GSH and several analogues has been shown to prevent alkylation of Cys-47 (22-24), consistent with the present observation. This interrelationship is interpreted to result from localized structural or conformational changes triggered by either event. Indeed, local conformational changes have been observed to result from site-directed mutagenesis at Cys-47 (25, 26) or replacement of Lys-54 (25), which forms an ion pair with Cys-47 in human π class GST. Based on the crystal structure of human π class GST complexed with S-hexylglutathione, 21.4 Å separates the sulfhydryl groups of Cys-47 in one subunit and Cys-101 in the other (5). The intersulfide distance within each subunit is about 18.3 Å. Molecular models indicate that the span between the two alkylating centers of chlorambucil cannot exceed 8.5 Å. Thus it is unlikely that the selective cross-linking we observe takes place on GST in that conformation. Our interpretation of these observations is that CLB is bound as a substrate at the hydrophobic substrate site (H-site) in the GSH-free dimer, as it is when GSH is present. The aromatic ring of chlorambucil is expected to enhance its binding affinity (27) in the apoenzyme, leaving the mustard side chains available for adventitious alkylation of nearby reactive nucleophiles. Affinity labeling by CLB has also been observed in its interaction with metallothionein (15) and requires that the rate of alkylation is slower than the rate of binding. The selective cross-linking observed with πGST provides additional evidence for conformational change in the absence of GSH and an estimation that the intersubunit distance between Cys-47 and Cys-101 in the dimeric apoenzyme is less than 8.5 Å. The concentration of πGST used is within the physiologic intracellular range (28). CLB was used with higher concentrations to optimize mass spectrometry analysis. The experimental results reported provide a mechanism for covalent drug sequestration and are consistent with the proposal (3, 28, 29) that this enzyme, whose synthesis is highly induced in patients receiving CLB and other chemotherapeutic agents (28), may serve itself as a trapping agent when GSH is depleted. Acknowledgment. The work was supported by a grant from the National Institutes of Health (GM-21248).

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Affinity Labeling of Apo-πGST (2) Mannervik, B., and Danielson, U. H. (1988) Glutathione transferasessstructure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283-337. (3) Tew, K. D. (1994) Glutathione-associated enzymes in anticancer drug resistance. Cancer Res. 54, 4313-4320. (4) Wilce, M. C. J., and Parker, M. W. (1994) Structure and function of glutathione S-transferases. Biochim. Biophys. Acta 1205, 1-18. (5) Reinemer, P., Dirr, H. W., Ladenstein, R., Huber, R., Lo Bello, M., Federeci, G., and Parker, M. W. (1992) Three-dimensional structure of class π glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 Å resolution. J. Mol. Biol. 277, 214-226. (6) Tamai, K., Satoh, K., Tsuchida, S., Hatayama, I., Maki, T., and Sato, K. (1990) Specific inactivation of glutathione S-transferase in class pi by SH-modifier. Biochem. Biophys. Res. Commun. 167, 331-338. (7) Ricci, G., Del Boccio, G., Pennelli, A., Lo Bello, M., Petruzzelli, R., Caccuri, A. M., Barra, D., and Federici, G. (1991) Redox forms of human placenta glutathione transferase. J. Biol. Chem. 266, 21409-21415. (8) Caccuri, A. M., Ricci, G., Desideri, A., Buffa, M., Fruttero, R., Gasco, A., and Ascenzi, P. (1994) Inhibition of human placenta glutathione transferase P1-1 by calvatic acid. Biochem. Mol. Biol. Int. 32, 819-829. (9) Jakobson, I., Askelo¨f, P., Warholm, M., and Mannervik, B. (1977) A steady-state-kinetic random mechanism for glutathione Stransferase A from rat liver. Eur. J. Biochem. 77, 253-262. (10) Ivanetich, K. M., and Goold, R. D. (1989) A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase. Biochim. Biophys. Acta 998, 7-13. (11) Nishihira, J., Ishibashi, T., Sakai, M., Tsuda, S., and Hikichi, K. (1993) Identification of the hydrophobic ligand-binding region in recombinant glutathione S-transferase P and its binding effect on the conformational state of the enzyme. Arch. Biochem. Biophys. 302, 128-133. (12) Dirr, H., Reinemer, P., and Huber, R. (1994) X-ray crystal structures of cytosolic glutathione S-transferases, implication for protein architecture, substrate recognition and catalytic function. Eur. J. Biochem. 220, 645-661. (13) Lo Bello, M., Pastore, A., Petruzzelli, R., Parker, M. W., Wilce, M. C. J., Federici, G., and Ricci, G. (1993) Conformational states of human placental glutathione transferase as probed by limited proteolysis. Biochem. Biophys. Res. Commun. 194, 804-810. (14) Yu, X., Wu, Z., and Fenselau, C. (1995) Covalent sequestration of melphalan by metallothionein and selective alkylation of cysteines. Biochemistry 34, 3377-3385. (15) Zaia, J., Jiang, L., Han, M. S., Tabb, J. R., Wu, Z., Fabris, D., and Fenselau, C. (1996) A binding site for chlorambucil on metallothionein. Biochemistry 35, 2830-2835. (16) Dulik, D. M., Colvin, O. M., and Fenselau, C. (1989) Characterization of glutathione conjugates of chlorambucil by fast atom bombardment and thermospray liquid chromatography mass spectrometry. Biomed. Environ. Mass Spectrom. 19, 248-252. (17) Morrison, J. R., Fidge, N. N., and Grego, H. (1990) Study on the formation, separation, and characterization of cyanogen bromide

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(18)

(19)

(20)

(21) (22)

(23)

(24)

(25)

(26)

(27)

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