Electron Paramagnetic Resonance Identification of a Highly Reactive Thiol Group in the Proximity of the Catalytic Site of Human Placenta Glutathione Transferase*

The reaction of the glutathione transferase from hu- man placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or pro- toporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration transferase GSH transferase EPR kinetics SI-maleimide; the GSH transferase activity as Titrations of the spin-labeled enzyme with hemin or protoporphyrin made at pH 7.0 by adding aliquots of the porphyrins to a solution of 65 p~ labeled GSH transferase. Liquid EPR recorded control system.

The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.
The GSH transferases (EC 2.5.1.18) are a family of isoenzymes that are known to initiate the biotransformation of toxic electrophiles by catalyzing their conjugation with glutathione (GSH) (1). In addition, most of these isoenzymes bind a wide spectrum of ligands and are supposed to act as intracellular carrier proteins for lipophilic endogenous metabolites and drugs (2). The mammalian GSH transferases include membrane-bound (3) and soluble isoenzymes, the latter accounting for more than 80% of the total cellular GSH transferase activity (4). The cytosolic isoenzymes are subdivided into three classes named a, p, and ?r characterized by different isoelectric points and catalytic and immunological properties (5). These isoenzymes are homo-or hetero dimers formed by reversible association of two polypeptide chains, * This work was supported by Consiglio Nazionale delle Ricerche Special Project "Biotecnologie e Biostrumentazione". The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. each containing an active site region (6). The human placenta GSH transferase, used for our study, belongs to the K class, is characterized by an acidic isoelectric point (PI = 4.6), and is composed of two identical subunits of molecular weight 22,500. Each subunit contains 209 amino acids and, in particular, 4 cysteines in the positions 14, 47, 101, and 169 (7). It has been reported that the activity of the GSH transferase from different sources is highly dependent on the integrity of at least 1 of the cysteinyl residues present in the protein (8)(9)(10)(11). We therefore decided to investigate the possible location of a thiol group at the active site of human placenta GSH transferase by making use of a nitroxide derivative of a thiol blocking reagent, namely maleimide (12). We found that the maleimide spin label can selectively react with a single thiol group per monomer. This group is important for the catalytic activity and must be located in the proximity of the lipophilic ligand and the substrate binding sites. Moreover, the different time-response of the maleimide binding as detected by EPR (2 min at pH 7.0), compared to the final enzyme inactivation (15 min), suggests a conformational rearrangement of the molecule between the two events. EXPERIMENTAL PROCEDURES sl-maleimide' and 4-amino-TEMPO were purchased from Aldrich, protoporphyrin IX and hemin from Fluka, S-methylglutathione and DTNB from Sigma. All other chemicals were of reagent grade.
The GSH transferase P was purified from human placentas by a slight modification of the procedure used for the human heart enzyme (13). The tissue, homogenized with 4 volumes of 10 mM potassium phosphate buffer, pH 7.0, containing 1 mM 2-mercaptoethanol, was subjected to one step of purification on a GSH affinity column (14) followed by chromatofocusing on an LKB fast protein liquid chromatography unit equipped with a Mono P HR 5/20 Pharmacia LKB Biotechnology Inc. column, as described elsewhere (13). The purity of the enzyme was tested by polyacrylamide gel electrophoresis in sodium dodecyl sulfate according to the method of Weber and Osborn (15). The GSH transferase activity was measured spectrophotometrically (340 nm) at 25 "C in 1 ml of 0.1 M potassium phosphate buffer, p H 6.5, containing 1 mM GSH and 1 mM l-chloro-2,4-dinitrobenzene (16); the resulting specific activity was 80 units/mg (1 enzymatic unit catalyzes the conjugation of 1 Fmol of substrate per min). The protein concentration was determined by the method of Bradford (17). Protein sulfhydryl groups were determined by the method of Ellman (18). Spin-labeled GSH transferase was prepared by incubation of 130 PM enzyme in 10 mM potassium phosphate buffer p H 7.0, with 50 excesses of sl-maleimide for 5 min a t 37 "C. The unreacted spin-labeling reagent was removed by dialyzing the sample against 10 mM potassium phosphate buffer, pH 7.0. The extent of spin label reacted with GSH transferase was estimated by double integration of the EPR signal before and after denaturation of the protein upon addition of concentrated NaOH. The resulting numerical value was compared to that obtained from a standard solution of 4-amino-TEMPO. The ' The abbreviations used are: sl-maleimide, N-(l-oxy1-2,2,6,6-tetramethyl-4-piperidiny1)maleimide; DTNB, 5-5'-dithiobis-(2-nitrobenzoic acid).

EPR Study of Human
Placenta GSH Transferase kinetics of the sl-maleimide binding were determined by adding slmaleimide at 1:l subunit/label ratio to GSH transferase samples buffered a t a known pH and recording an EPR spectrum every 50 s. The samples at pH 6.0 and 7.0 were prepared by dialyzing aliquots of 100 p M GSH transferase against 10 mM potassium phosphate buffer, pH 6.0 or 7.0, respectively. After dialysis, the GSH transferase was diluted to the appropriate concentration for the EPR measurement, usually 40 p~. The sample a t p H 8.0 was prepared by addition, just before the EPR experiments, of diluted KOH to a GSH transferase solution at pH 7.0, to avoid as much as possible the oxidation of the protein thiol groups. The kinetics of binding in presence of Smethylglutathione or protoporphyrin IX were determined after adding 2 eq of SI-maleimide to a GSH transferase solution buffered a t pH 7.0 and incubated for 5 min a t 25 "C with the appropriate concentration of the ligand. The kinetics of the enzyme inactivation was followed by taking, a t different times, aliquots of 5 4 from a 10 mM potassium phosphate buffer pH 7.0 solution containing 1.24 p M GSH transferase and 2 eq of SI-maleimide; the GSH transferase activity was measured as described above. Titrations of the spinlabeled enzyme with hemin or protoporphyrin IX were made at pH 7.0 by adding aliquots of the porphyrins to a solution of 65 p~ labeled GSH transferase.
EPR spectra were run on an ESP 300 Bruker spectrometer operating at 9 GHz. Temperature was maintained at 25 "C with a Bruker constant temperature device. The sample was contained in a glass capillary with an internal diameter of 1.10 mm. Liquid nitrogen EPR spectra were recorded using the Bruker temperature control system.

RESULTS
The EPR spectrum of the GSH transferase incubated for 5 min at 37 "C with a 50-fold molar excess of sl-maleimide and then dialyzed against 10 mM potassium phosphate buffer, pH 7.0, to remove the unreacted compound, is shown in Fig. lA. The spectrum is mainly characterized by a strongly immobilized nitroxide species ( 2 A l l = 64 G) and by a small component accounting for less than 5% of the total signal and characteristic of free nitroxide (Fig. 1B) not completely removed by dialysis. Double integration of the EPR signal indicates 2 mol of nitroxide label bound per mol of enzyme.
An EPR spectrum with identical line shape could be obtained simply by adding to the enzyme 2 eq of maleimide spin label. In this condition, the kinetics of the binding can be followed easily, taking advantage of the fact that the free and less than 2 min with 50% inhibition of activity, while the second one is completed in about 15 min with a final enzyme inhibition of 80%. Titration of the SH groups of the protein with DTNB, before incubation with sl-maleimide, revealed the presence of four reactable sulfhydryls. On the other hand, after the first 2 min of reaction with sl-maleimide, only two S H groups react with DTNB. They remain reactive after 20 min or more. A plot of the disappearance of the free label EPR signal against time is also reported in the inset of Fig. 2. The kinetics of binding of sl-maleimide has also been measured at pH 6.0. In this case, the rate of disappearance of the free label is much slower, which allowed us to better understand the mechanism of binding. The kinetics of binding at pH 6.0 a t 1:l subunit/label ratio is reported in Fig. 3.
T o gain information about the structural location of the 2 cysteine residues, the kinetics of the binding of the sl-maleimide were also followed at pH 7.0 in the presence of two compounds known to inhibit the activity of the GSH transferase (I), namely S-methylglutathione and protoporphyrin IX. The kinetics, reported in Fig. 4, indicate in both cases a decrease of the rate of reaction of the sl-maleimide. As a final experiment, a titration with either the paramagnetic hemin or the diamagnetic protoporphyrin IX on the sl-maleimide- The EPR intensity of the spectra presented in Fig. 5 , evaluated by double integration, is reported as a function of hemin concentration (m). The EPR intensities for the corresponding titration with the diamagnetic protoporphyrin IX are also shown for comparison (0). reacted enzyme was carried out. Protoporphyrin IX was found not to have any effect, either on the shape or on the intensity of the nitroxide EPR signal (results not shown), while hemin produced a decrease of the EPR intensity without any perturbation of the line shape (Fig. 5 ) . A plot of the intensity of the EPR signal, evaluated by double integration, against the ligand concentration is reported in Fig. 6. The EPR signal of iron from the hemin was observed a t liquid nitrogen temperature during the same titration. The signal, due to a high spin iron(II1) with axial symmetry, is not perturbed by the presence of the protein suggesting that the heme-protein interaction does not happen through the metal but likely through the porphyrin ring.

DISCUSSION
The EPR spectrum of Fig. L4 is characterized, apart from a free label component accounting for less than 5% of the total signal, by a single bound species ( 2 A l 1 = 64 G) which accounts for 2 mol of sl-maleimide reacted per mol of enzyme. In parallel, two sulfhydryl groups disappear as determined by titration with DTNB. The EPR signal of the bound spin-label is homogeneous and has a line shape completely identical with that displayed by enzyme which was reacted with 1 eq of spin-label reagent (spectrum not shown). This indicates that the reaction occurs with two identical thiol groups (i.e. one for each monomer) that confer the same physicochemical constraints on the label. The two thiols are very reactive, in fact, at pH 8.0, 2 eq of sl-maleimide can bind to the protein in less than 50 s. The reaction is also very fast at pH 7.0 (Fig.  2, inset), while the kinetics of inactivation of the enzyme displays a different time course (Fig. 2). In fact, two distinct phases can be detected; the first one with an inhibition of 50% ends in less than 2 min, a time that corresponds to that required for binding 2 eq of sl-maleimide at the same pH as detected by EPR. The second phase is much slower: it ends up in 15 min and reduces further the residual enzymatic activity. A likely explanation is that after the fast binding of sl-maleimide to the thiol groups, a slow rearrangement of the protein takes place to reach an almost inactive conformation. In line with this is the observation that after 2 min only two sulfhydryl groups are titrable with DTNB. The drastic inactivation (50%) observed after 2 min (Fig. 2), the time required for the binding of sl-maleimide, as detected by EPR, suggests a close proximity between the thiol group and the active site of the enzyme. This is in agreement with the experimental finding that the GSH transferase activity is strongly dependent on the integrity of the SH groups (8)(9)(10)(11) and with the recent result that alkylation of this enzyme with a fluorescent maleimide probe gives rise to enzyme inactivation and selective modification of the Cys-47 residue (19). The binding reaction between the sulfhydryl groups and the sl-maleimide has been more thoroughly studied at pH 6.0 by taking advantage of the slower rate displayed by the reaction at this pH. The reaction kinetics are second order, with a calculated rate constant evaluated from the slope of the line of Fig. 3 of 110 & 1 M" s" which is in agreement with the value obtained by plotting the pseudo-first order constants against enzyme concentration (data not shown).
Information about the location of the reacting thiol group comes from the kinetics of binding of the sl-maleimide in the presence of S-methylglutathione (Fig. 4). The reaction is slowed down with respect to that shown by the native enzyme, suggesting that this group is located near the GSH binding site (G site) (6), or that the binding of S-methylglutathione produces a conformational change able to screen the accessibility of such a group. A similar behavior is also displayed by the kinetics of disappearance of the free sl-maleimide in the presence of an excess of protoporphyrin IX (Fig. 4). The rate of binding decreases with respect to that obtained in the absence of protoporphyrin, again suggesting a close proximity between the thiol group and the heme binding site. The evidence of a close proximity between the porphyrin binding site and the reacting SH group also comes from the experiments reported in Fig. 6, where a plot of the EPR intensity of the sl-maleimide-reacted enzyme is reported as a function EPR Study of Human Placenta GSH Transferase of the paramagnetic hemin or the diamagnetic protoporphyrin IX concentrations. The reduction of the label signal intensity is evident only for the titration with hemin and can be interpreted as due to a dipole-dipole interaction occurring between the maleimide nitroxyl group and the iron. The theory to describe the line shape of an EPR signal which is influenced by dipolar coupling to a second spin in a rigid arrangement has been developed by Leigh (20) and has been applied to several systems. According to this theory, the distance between the spin label moiety and the Fe3+ atom of the heme bound to the enzyme is estimated from the magnitude of the interaction coefficient C, which may be obtained from the measured fractional diminution in the amplitude of the EPR signal and the measured line width of the signal in the absence of paramagnetic interaction. The interaction coefficient C is related to interspin distance by the equation: where g is the electronic g factor of the observed spin, p is the magnetic moment of the paramagnetic ion, 7 is the correlation time for the modulation of the dipolar interaction, and r is the distance between Fe3+ and the spin label. Because the iron of the hemin bound to the enzyme had the usual axial high spin EPR spectrum displayed by hemin, a correlation time ranging between 1 0 " ' -and lo-" (21) was taken for the iron from which a label-iron distance of 10 A s r s 14 A was obtained. The highly reactive thiol group important for activity must then be located in between or across the hydrophobic ligand and the substrate binding sites in agreement with that found in rat liver ligandin (8) and in horse GSH transferase (11). Moreover, this group is characterized by a low pK, and because of its proximity to the active site, this could be determined by the same environment that induces a lowering of the pK of the enzyme-bound GSH in the active site (22). In addition, the kinetics of the enzyme inactivation observed after the binding of sl-maleimide indicates that the cysteine integrity plays a crucial role for the maintainance of a catalyticaly active enzyme conformation.