©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Human Glutathione Transferase P1-1
SPECTRAL, KINETIC, AND STRUCTURAL PROPERTIES OF Cys-47 AND Lys-54 MUTANTS (*)

(Received for publication, August 10, 1994; and in revised form, October 31, 1994)

Mario Lo Bello (1) Andrea Battistoni (1) Anna P. Mazzetti (1) Philip G. Board (2) Masami Muramatsu (3) Giorgio Federici (1) Giorgio Ricci (1)(§)

From the  (1)Department of Biology, University of Rome ``Tor Vergata,'' 00133 Rome, Italy, (2)The John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia, and the (3)Department of Biochemistry, Saitama Medical School, Moroyama, Iruma, Saitama 350-04, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the human placental glutathione transferase, Cys-47 possesses, at physiological pH values, a pKvalue of 4.2 and may exist as an ion pair with the protonated -amino group of Lys-54. Using site-directed mutagenesis we investigate spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. The results shown indicate that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47. The contribution of Lys-54 to the activation of Cys-47 is assessed by the spectral properties of the K54A mutant enzyme. The induced cooperativity toward glutathione, as a consequence of mutation of Lys-54 to alanine, clearly parallels that observed for the Cys-47 mutant enzymes (see the preceding paper (Ricci, G., Lo Bello, M., Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W., and Federici, G.(1995) J. Biol. Chem. 270, 1243-1248) and points out the importance of this electrostatic interaction in shaping the correct spatial arrangement for the binding of glutathione and in anchoring the flexible helix alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity.


INTRODUCTION

The glutathione transferases (EC 2.5.1.18) (GST) (^1)are a family of enzymes involved in the mechanism of cellular detoxification. They catalyze the nucleophilic attack of glutathione on the electrophilic center of a number of toxic compounds and xenobiotics(1) . The cytosolic enzymes have been grouped into four species-independent classes: Alpha, Mu, Pi, and Theta, on the basis of N-terminal sequence, substrate specificity and immunological properties(2, 3) .

Human placental glutathione transferase (Class Pi) (GST P1-1)(4) , a homodimeric protein of about 46 kDa, has been extensively studied in different laboratories because of the clinical interest in it as a potential marker during chemical carcinogenesis (5, 6) and its potential role in the mechanism of cellular multidrug resistance against a number of antineoplastic agents(7, 8, 9) . Although a number of amino acid residues involved in the binding of GSH have been identified by crystallographic analysis (10) and site-directed mutagenesis studies (11, 12, 13) , the catalytic mechanism is not completely clarified. Moreover, the key structural role of some residues is still unknown. The function of cysteinyl residues has been extensively investigated (14, 15) , leading to the conclusion that none of these residues is involved in the catalysis. However, the surprising results that mutation of Cys-47 in alanine or serine induces a positive cooperativity between the two subunits (see the preceding paper(29) ) shed more light on the importance of this residue and prompted us to investigate further its structural role. We previously suggested that this residue exists as an ion pair with the protonated -amino group of Lys-54 (16) and its thiolate form is characterized by a sharp UV absorption spectrum centered at 229 nm. However, spectral and kinetic experiments together with electrostatic calculations gave us only indirect evidence of the ion pair existence. In this paper we investigate, by site-directed mutagenesis, the role of both these residues providing answers to questions such as: 1) given the fact that there are two reactive cysteines (Cys-47 and Cys-101) can the spectral band centered at 229 nm be assigned exclusively to Cys-47? 2) Is Lys-54 the only positive charge responsible for the activation of Cys-47? 3) If the cooperativity induced by mutation of Cys-47 is a consequence of disruption of the ion pair, does the mutation of Lys-54 (the counter part of the ion pair) also causes positive cooperativity? To this aim we have replaced Lys-54 with Ala to neutralize the side chain positive charge of this residue. This mutant enzyme was expressed in Escherichia coli, purified and its kinetic and spectral properties investigated by comparison with the wild type and the cysteine mutant enzymes. The results shown in this paper suggest that the thiolate spectral band should be assigned exclusively to Cys-47 and that mutation of Lys-54 causes a remarkable decrease of the thiolate spectral band and also induces positive cooperativity. Limited proteolysis experiments with trypsin were also carried out on all these mutant enzymes to probe for conformational changes upon replacement of either of the residues of this ion pair. Finally, heat inactivation studies were performed on the same mutants to assess whether the induced cooperativity was achieved at the expense of some loss of thermal stability of the protein.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids

The E. coli strains BMH 71/18 mutS (17) and BMH 71/18 (18) were from our own collection, whereas the strain Top 10 was from Invitrogen. Plasmid pGST-1, expressing human GST P1-1 in the cytoplasm of E. coli under control of the inducible promoter ptrc has been described elsewhere. (^2)Plasmid p18Seq-1 was obtained inserting a 780-base pair SphI-SphI DNA fragment from plasmid pGST-1 in the SphI site of plasmid pEMBL 18(+)(20) ; this plasmid contains the entire sequence coding for GST P1-1 from human placenta (5) less the first 55 nucleotides.

Site-directed Mutagenesis

Site-directed Mutagenesis of Cysteinyl Residues (Cys-47 and Cys-101)

Plasmid p18seq-1 was used to generate single-stranded DNA template to be used in oligonucleotide-directed mutagenesis experiments. The following oligonucleotides were used: Ala-47, 5`-AAAGCCTCCGCCCTATACGG; Ser-47, 5`-AAAGCCTCCTCCCTATACGG; Ala-101, 5`-GACCTCCGCGCCAAATACAT; Ser-101, 5`-GACCTCCGCTCCAAATACAT. Mutagenesis reactions were performed as follows: 0.5 pmol of single-stranded DNA were mixed with 5 pmol of the mutagenic oligonucleotide in 20 µl of 20 mM Tris-HCl, pH 7.5, 2 mM MgCl(2), 50 mM NaCl, incubated for 2 min at 80 °C, and allowed to cool slowly at room temperature. This hybridization mixture was added to 80 µl of 20 mM Tris-HCl, pH 7.5, 2.5 mM DTT, 12,5 mM MgCl(2), 62.5 µM each of dATP, dTTP, dCTP, dGTP, and 0.05 mM ATP, and the in vitro DNA polymerization was started by the addition of 1 unit of native T7 DNA polymerase and 2 units of T4 DNA ligase. The reaction mixtures were incubated for 1 h at 37 °C and then terminated by the addition of EDTA (15 mM as final concentration). Competent BMH 71/18 mutS cells were transformed with the mutagenesis mixtures, inoculated in 10 ml of LB containing 100 µg/ml ampicillin, and grown overnight at 37 °C. In order to obtained segregation of mutant and wild type plasmids DNA was extracted and transformed in BMH 71/18 that were plated on LB-ampicillin. Colonies containing the mutated plasmid were identified by colony hybridization, using the radiolabeled mutagenic oligonucleotides as hybridization probes. Nucleotide sequence analysis was carried out by the chain termination method. The mutated 780-base pair SphI-SphI DNA fragments from plasmid p18 seq1 were newly subcloned in the expression plasmid pGST-1 to produce the mutant enzymes.

Site-directed Mutagenesis of Lys-54

In the case of K54A mutant enzyme the GST P1-1 M13 mp18 mutagenesis template described by Board et al.(21) and an Amersham Kit were used with the oligonucleotide 5`-GTC CTG GAA CGC GGG GAG CTG to create a K54A substitution by site-directed mutagenesis. M13 clones containing the required mutation were identified by DNA sequencing. The cDNA insert containing the mutation was excised by SphI-HindIII digestion and ligated into a gel purifed SphI-HindIII cut pRB307 vector(22) .

Expression and Purification of Mutant and Wild Type GST P1-1 Enzymes

Expression of Wild Type and Cysteine Mutant Enzymes (C47A, C47S, C101A, C101S, C47S/C101S, C47A/C101A)

Expression experiments were performed using the E. coli strain Top 10.^2 Single colonies of freshly plated bacteria were used to inoculate 5-ml overnight cultures. These cultures were diluted 1:40 into LB medium containing 100 µg/ml ampicillin, grown at 37 °C to an A value of 0.5 and induced by the addition of 2 mM isopropyl-beta-D-thiogalactopyranoside. Cell were grown at 37 °C for 18 h, harvested by centrifugation for 15 min at 7000 times g, resuspended in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 10 mM DTT. Cells were incubated on ice for 30 min in the presence of 0.25 mg/ml lysozyme and subsequently disrupted by sonication. Cell debris were removed by centrifugation at 20,000 times g for 25 min, and the resulting supernatant was centrifuged at 100,000 times g for 60 min.

Expression of Lysine Mutant Enzyme (K54A)

E. coli expressing GST P1-1 with the K54A mutation in the ubiquitin fusion vector pRB307 (22) were grown in the presence of 0.1 mM isopropyl-beta-D-thiogalactopyranoside and were processed as previously described by Board and Pierce(23) .

Wild type and mutant enzymes were purified by affinity chromatography on immobilized glutathione(24) .

Analysis of Purified Proteins

Protein concentration was determined by the method of Lowry et al.(25) . The enzymatic activity was measured at 25 °C according to the method of Habig and Jakoby(26) . SDS-PAGE was carried out by the method of Laemmli (27) with 12% (w/v) polyacrylamide resolving gels. HPLC analysis was performed on a Vydac 201 TP54 300 Å reverse-phase C18 column, Vydac (Hesperia CA, U.S.A.) using acetonitrile and water, both containing 0.1% trifluoroacetic acid, as eluting solvents(28) . A gradient from 35% to 55% was achieved in 24 min, followed by a 5 min gradient of 55-65% acetonitrile.

After affinity purification the wild type and the mutant enzymes were all homogeneous as judged by reverse-phase HPLC and SDS-PAGE. The retention time in the HPLC system and the apparent subunit molecular weight (about 23 kDa) were similar to those of the wild type enzyme. The kinetic parameters of the K54A mutant enzyme are reported in Table 1.



GST Activity and Kinetic Studies

Spectrophotometric assay for GST activity and kinetic analyses were performed as previously described(29) .

Spectrophotometric Determination of Thiol Groups

Protein sulfhydryl groups were titrated essentially by following the Ellman (30) procedure; about 100 µg of wild type or mutant enzymes were reacted with 0.5 mM 5,5`-dithiobis-(2-nitrobenzoic acid) in 1 ml final volume of 0.1 M phosphate buffer, pH 8.0. The reactions were continuously followed spectrophotometrically at 412 nm using A = 13,600 M cm. Among the three titratable cysteines (31) Cys-47 and Cys-101 are both accessible to the solvent and display high reactivity for 5,5`-dithiobis-(2-nitrobenzoic acid), at pH 8.0. The third residue, which titrated slowly, could be Cys-169, since Cys-14 is deeply buried in the protein structure (10) (data not shown).

UV Difference Spectra

UV difference spectra of wild type and mutant enzymes were obtained as described previously(16) . Briefly, sample and reference cuvettes contained 1 ml of GST P1-1 or mutant enzymes solution (final concentration 0.1 mg/ml; 4.4 µM Cys-47) in 0.1 M buffer at suitable pH values. The reaction was started by adding 10 µl of a solution containing 17.6 nmol of bromopyruvate in the sample and 10 µl of buffer in the reference cuvette. At fixed times spectra were recorded and aliquots were withdrawn for assaying enzymatic activity.

Limited Proteolysis

Limited proteolysis of wild type or mutant enzymes was carried out as described previously (31) using trypsin as proteolytic agent (10%, w/w) at 37 °C in 0.1 M ammonium bicarbonate, pH 7.8. At different times aliquots were withdrawn from the mixture and assayed for GST activity. The inactivation rates of all the mutant enzymes were calculated in the linear part of the time course reaction. At the same times, aliquots (about 10 µg of protein) were diluted 2-fold with sample buffer, boiled at 100 °C for 10 min, and subjected to SDS-PAGE in a Tricine SDS-discontinuous buffer system according to Schagger and Von Jagow(32) . After electrophoresis protein bands were stained with Coomassie Blue. In the case of K54A mutant enzyme, the experiments were carried out under the same conditions, except a lower temperature (25 °C) was used due to a spontaneous loss of activity of this mutant enzyme at 37 °C.

Thermal Stability

The wild type and the mutant enzymes were previously incubated with 0.1 M DTT in 0.01 M phosphate buffer at pH 7.0, at 37 °C, and subsequently subjected to gel filtration on Sephadex G-25 to eliminate excess DTT. All these samples were incubated at each temperature for at least 30 min, at protein concentration of 0.1 mg/ml in 0.1 M phosphate buffer, pH 6.5, containing 0.1 mM EDTA. At each time aliquots were withdrawn from the incubation mixture and assayed for GST activity.


RESULTS

UV Difference Spectra of Cys-47 and Cys-101 Mutant Enzymes

UV difference spectra of cysteine-mutant enzymes after reaction with bromopyruvate are shown in Fig. 1. Among the mutant enzymes, C101A displays a similar spectral band centered at 229 nm as the wild type, whereas C47A and C47A/C101A (both containing the mutated Cys-47) do not have any spectral perturbation at the same wavelength. Replacement of Cys-47 and Cys-101 with serine gave similar spectrophotometric results (data not shown). Thus, it is concluded that the spectral band should be assigned exclusively to the thiolate ion of Cys-47. Reaction of wild type and C101A, C47A, C47A/C101A mutant enzymes with bromopyruvate gives rise to different inactivation patterns (Fig. 1). The alkylation of wild type and C101A mutant yields rapid and complete loss of activity (Fig. 1, A and B). Also C47A mutant undergoes a considerable inactivation by reaction with bromopyruvate, but after 20 min the residual activity is about 20% and does not change within 100 min. Similar partial inactivation has been observed for the C47A mutant enzyme of the rat GST P reacted with cystamine(14) . Therefore, chemical modification of C47A mutant may induce some structural perturbation that affects also catalytic activity. In the case of the double mutant Ala 47/101, there is a partial loss of activity but the inactivation kinetics is much slower than that reported for the wild type or Cys-47 and Cys-101 mutant enzymes. This result indicates that the alkylation of the third residue (possibly Cys-169(33) ), located in the second domain and very far from the active site may still influence the enzymatic activity.


Figure 1: UV difference spectra of wild type and cysteine-mutant enzymes after reaction with bromopyruvate. 2.2 µM wild type (4.4 µM Cys-47) or cysteine mutant enzymes in 0.1 M potassium phosphate buffer, pH 7.0, were placed in the sample and the reference cuvettes and 17.6 µM (final concentration) bromopyruvate was added in the sample compartment. A, wild type (curves a, b, and c: time 0, 1, and 15 min, respectively); B, C101A mutant (curves a, b, and c: time 0, 1, and 20 min, respectively); C, C47A mutant (curves a, b, and c: time 0, 4, and 120 min, respectively); D, C47A/C101A mutant (curves a, b, and c: time 0, 3, and 120 min, respectively). Inset, time course of inactivation.



Spectral Properties of K54A Mutant Enzyme

UV difference spectra recorded during the reaction of bromopyruvate, at pH 7.0, with K54A mutant enzyme clearly indicate that the spectral band centered at 229 nm was dramatically reduced to about 25% of that recorded in the wild type enzyme, under the same conditions. (Fig. 2). The pH dependence of this residual spectral band at 229 nm is consistent with a shift of a pK(a) value of the reactive thiol from 4.2 to 5.3. Thus, replacement of the positive charge of Lys-54 with the neutral Ala residue strongly affects the extent of the ion pair formation. At pH 7.0, the reaction with bromopyruvate is completed within 15 min, concomitant with a full enzymatic inactivation (data not shown). The fact that in the K54A mutant enzyme the reactivity of this thiol is still elevated and the spectral band centered at 229 nm does not completely disappear may indicate that other electrostatic interactions stabilize the negative charge of Cys-47, or, alternatively, that a different rearrangement of the region surrounding the Cys-47 in the K54A mutant enzyme could facilitate an interaction with a different positive charged residue. In this respect it has been previously demonstrated (31) that Cys-47 is located in a highly mobile region responsible for conformational changes upon GSH binding to the active site. It is possible that in K54A mutant enzyme this region becomes more flexible and in this way Cys-47, in the absence of Lys-54, may establish a weak ion pair with an other positive charge by approaching it during its motion. Whatever it is, Lys-54 residue seems to be important in promoting formation of thiolate, but we need to consider other important ionic interactions, which are, at the moment, unknown.


Figure 2: pH dependence of the apparent molar extinction of the spectral band at 229 nm. 2.2 µM of wild type or K54A mutant enzymes were placed in the sample and reference cuvettes in the presence of 0.1 M buffer at suitable pH values. The reaction was started by addition of 8.8 µM bromopyruvate in the sample. The apparent values were calculated at the end of the reaction, when both the absorbance at 229 nm, and the activity values became constant. bullet, wild type; circle, K54A mutant enzyme.



Homotropic Behavior of K54A Mutant Enzyme

Since Cys-47-mutant enzymes have been shown to possess an induced cooperativity toward glutathione (the preceding paper(29) ), one would think that a similar behavior may be induced by replacement of Lys-54, the counter part of ion pair with Cys-47. The K54A mutant enzyme exhibits sigmoidal GSH saturation kinetics (Fig. 3). As in the case of Cys-47 mutant enzyme the data fit well to a rate equation expressing positive cooperativity between the two G sites(29) . The Hill coefficient, measured as previously reported, was 1.4, a value very similar to that found for the single Cys-47 mutant enzymes(29) . On the other hand, no homotropic or heterotropic behavior has been observed for CDNB, the second substrate (data not shown). These results together with those shown in the preceding paper present a strong evidence that disruption of the ion pair by mutation of either residue (Cys or Lys) is the basis for the observed induced cooperativity.


Figure 3: Homotropic behavior of K54A mutant enzyme. GSH saturation curve of the K54A mutant enzyme at 25 °C was obtained as described under ``Experimental Procedures.'' Initial velocities are expressed as DeltaA/min. GSH concentration is expressed as mM. The data were fitted by nonlinear least squares regression analysis with Equation 1 previously described(29) . The line of best fit is shown. Inset, Hill plot of data. n(H) value was 1.4.



Limited Proteolysis of Mutant Enzymes

Limited proteolysis has been used to probe for conformational changes of GST P1-1(31) . Digestion with trypsin, in the absence of glutathione, was effective on all the mutant enzymes giving a proteolytic pattern (analyzed by SDS-PAGE) very similar to that of wild type (data not shown). The susceptibility to proteolytic attack was greatly increased in the case of mutant enzymes where Cys-47 was replaced with serine or Lys-54 with alanine, respectively. The rates of proteolysis, calculated as described under ``Experimental Procedures'', were 180%, 240% and 380% for C47S/C101S, C47S and K54A, respectively as compared to that of wild type. Conversely, Cys-101 mutant enzyme showed a velocity of proteolysis similar to that of wild type. Since serine is considered to be isosterical to cysteine, it is evident that the only difference between them is the negative charge that Cys-47 possesses as a thiolate ion. This finding suggests that destruction of the ion pair makes the flexible region (helix alpha 2) more exposed to the solvent than in wild type or C101S mutant enzymes.

Thermostability Studies

On the basis of previous results indicating that mutation of either residue of the ion pair may induce a different spatial arrangement of the region close to the G-site, we studied the thermal stabilities of wild type and all the mutant enzymes. From Fig. 4it can be seen that wild type and the Cys mutant enzymes were substantially stable over the range of temperature: 25-45 °C, whereas K54A mutant enzyme underwent, in the same range, a consistent inactivation. Remaining activities after 10-min incubation showed that the mid point of the temperature stability curve was 52 °C for the wild type and 40 °C for K54A. Therefore, replacement of Lys-54 with Ala may induce a temperature-dependent irreversible conformational change, whereas replacement of Cys-47 and Cys-101 with Ala or Ser, respectively, does not change the thermostability properties of GST P1-1. These experiments suggest that the heat inactivation, observed in the K54A mutant, is not related to the cooperativity conferred by mutating either residue (Cys-47 or Lys-54) of the ion pair but it may reflect an additional and different structural role for Lys-54. This residue, conserved in all the known sequences of the Pi class GST(34) , is situated at the beginning of the strand beta 3, close to Asp-57 a residue which has been reported to play a structural role(35) . Since both residues belong to the same beta-strand (strand beta 3) it is possible that this region could be involved in the maintenance of the proper stable conformation of the enzyme.


Figure 4: Thermal stability of the wild type and mutant enzymes. The enzyme was subjected to heat inactivation studies as described under ``Experimental Procedures.'' The remaining activity was assayed after 10 min of incubation at different temperature values. , wild type; Delta, C47A mutant; asterisk, C101A mutant; box, C47A/C101A mutant; , C47S mutant; circle, C47S/C101S mutant; bullet, K54A mutant.




DISCUSSION

Chemical modification studies on GST P1-1 using thiol reagents suggested that Cys-47 is the likely target of this reaction causing a fast inactivation process(36, 37, 38) , even though this residue is not involved in the catalytic mechanism(14, 15) . The possible reason for this finding may rely on the particular position of this residue which is situated at the end of the flexible helix alpha 2, close to the G-site. Crystallographic analysis of the GST P1-1, in complex with S-hexylglutathione, reveals that Cys-47 is located on the surface with its thiol group pointing into a small hydrophobic pocket, formed by some main chain atoms of Lys-44 and Gln-51 and the side chain atoms of Trp-38 and Leu-52. Since the same residues are involved, on the opposite side, in the binding of GSH one could explain why chemical modification of Cys-47 disturbs the binding of GSH, whereas the presence of this substrate renders Cys-47 less accessible to the solvent(39) . Thus, Cys-47 may act as a molecular switch for different conformations, depending whether GSH or thiol-reagents bind first to the enzyme. A very similar behavior has been observed for Cys-289, a residue close to -Glu-Cys site in the glutathione synthetase from E. coli(40) . Thus, the role played by Cys-47 in GST P1-1 could be also of more general interest for different proteins.

Using site-directed mutagenesis we have demonstrated that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47 (see Fig. 1) despite of the fact that Cys-101 displays high reactivity and its modification inactivates the enzyme. The existence of the ion pair with Lys-54 is suggested from the substantial decrease of value at 229 nm and the shift in the pK value from 4.2 to 5.3 for the thiol group, observed in the K54A mutant enzyme (see Fig. 2). These results need to consider other important electrostatic interactions in lowering the pK value of Cys-47, in the case of K54A mutant. One possibility is that the flexible helix alpha 2, where Cys-47 is located, may undergo an increased motion by approaching the thiol group to on other positive charge. As an example, Lys-44 is 7.9 Å from the sulfur atom of Cys-47 in the three-dimensional structure complexed with S-hexylglutathione, but this distance could be substantially shortened during this motion. Therefore, Lys-44 could be the putative residue which contributes to the formation of fluctuating ion pair, in the absence of Lys-54. The hypothesis suggesting structural changes is corroborated by experiments employing limited proteolysis which demonstrate that replacement of Lys-54 with alanine leads to an increased proteolytic susceptibility.

The homotropic behavior found for the K54A mutant enzyme (see Fig. 3) clearly parallels the positive cooperativity shown by the Cys-47 mutant enzymes. It is evident that the electrostatic interaction (Cys-47-Lys-54) is important in shaping the correct spatial arrangement for the binding of GSH to the active site and in anchoring the flexible helix alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity. To determine if this novel phenomenon leads to some change in the thermal stability of GST P1-1, heat inactivation experiments were carried out with the wild type and the mutant enzymes. The results clearly indicate that the cooperativity acquired by mutation of Cys-47 residue does not change the thermal stability properties shown by the wild type enzyme. In contrast, mutation of Lys-54 causes a lose of catalytic activity at a substantially lower temperature (see Fig. 4). It may therefore be concluded that while the kinetic parameters proteolytic experiments, and induced cooperativity of Cys-47 and Lys-54 mutant enzymes indicate a rearrangement of the helix alpha 2 due to disruption of the ion pair, thermal stability properties are not related to the existence of this electrostatic interaction and may reflect an additional structural role for Lys-54.

As an hypothetical structural explanation, we can speculate from inspection of the three-dimensional structure of GST P1-1 (see Fig. 5) as follows: mutation of Cys-47 leads to structural perturbation in the G-site resulting in a reduced affinity for glutathione. The perturbation is also transmitted to the other monomer, possibly via helix 4 which consists of residues involved in subunit contacts (Asp-98, Asp-94, Met-91) and residues implicated in the binding and catalysis of substrate (Tyr-108, Ile-104, Cys-101, Asp-98). Residues Tyr-49 and Gln-64 are also implicated in the transmission, because they are located close to Cys-47 and are also involved in subunit contacts with helix 4 of the other monomer. The binding of glutathione appears sufficient to correct the perturbations in helix 4 due to the Cys-47 mutation, leading to return of the high affinity binding site in the second monomer. Crystallographic studies of the Cys-47 and Lys-54 mutant enzymes will clarify structural transitions coupled with local structural changes during the allosteric transitions.


Figure 5: Ribbon representation of the human glutathione transferase P1-1 showing potential residues involved in the observed positive cooperativity (see text). The view is taken down the 2-fold axis of the dimer molecule. The figure is based on the crystal structure of the enzyme in complex with S-hexylglutathione (10) (the inhibitor is denoted by hexyl and GSH in the figure) and was produced by the computer program MOLSCRIPT(19) .




FOOTNOTES

*
This work was partially supported by grants from Consiglio Nazionale delle Ricerche, Progetto Finalizzato: ``Applicazioni cliniche della ricerca oncologica,'' and Progetto Finalizzato ``Biotecnologie e Biostrumentazione.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biology, University of Rome, ``Tor Vergata,'' Via della Ricerca Scientifica, 00133 Rome, Italy. Tel.: 06-72594375; Fax: 06-2025450.

(^1)
The abbreviations used are: GST, glutathione transferase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; LB, Luria-Bertani medium; mutated enzymes are designated using one-letter code for the original amino acid (before the residue number) and for the replaced amino acid after the residue number.

(^2)
A. Battistoni, P. Mazzetti, R. Petruzzelli, M. Muramatsu, G. Ricci, G. Federici, and M. Lo Bello, submitted for publication.


ACKNOWLEDGEMENTS

We thank Prof. Andrea Bellelli for helpful discussion.


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