Probing Subunit Interactions in Alpha Class Rat Liver Glutathione S-Transferase with the Photoaffinity Label Glutathionyl S-[4-(Succinimidyl)benzophenone]
2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês
10.1074/jbc.275.8.5493
ISSN1083-351X
AutoresJibo Wang, Susanne Bauman, Roberta F. Colman,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoGlutathionylS-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathioneS-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K R) of 99 ± 2 μm andk max of 0.082 ± 0.005 min−1. Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Δ5-androstene-3,17-dione and 17β-estradiol-3,17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2. GlutathionylS-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathioneS-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K R) of 99 ± 2 μm andk max of 0.082 ± 0.005 min−1. Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Δ5-androstene-3,17-dione and 17β-estradiol-3,17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2. glutathioneS-transferase 1-chloro-2,4-dinitrobenzene glutathionyl S-[4-(succinimidyl)benzophenone] monobromobimane matrix-assisted laser desorption ionization time-of-flight ethacrynic acid Δ5-androstene-3,17-dione Protein Data Bank high pressure liquid chromatography Glutathione S-transferases (GST)1 (EC 2.5.1.18) are a family of detoxification enzymes that catalyze the conjugation reaction of glutathione with a variety of endogenous electrophiles and xenobiotics (1.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (954) Google Scholar, 2.Mannervik B. Danielson U.H. CRC Crit. Rev. Biochem. 1988; 23: 283-337Crossref PubMed Scopus (1669) Google Scholar, 3.Rushmore T.H. Pickett C.B. J. Biol. Chem. 1993; 268: 11475-11478Abstract Full Text PDF PubMed Google Scholar, 4.Wilce M.C.J. Parker M.W. Biochim. Biophys. Acta. 1994; 1205: 1-18Crossref PubMed Scopus (538) Google Scholar). The enzyme catalyzes the reaction by lowering the pK a of the sulfhydryl group of the enzyme-bound glutathione (5.Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. Armstrong R.N. Ji X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (410) Google Scholar, 6.Wang R.W. Newton D.J. Johnson A.R. Pickett C.B. Lu A.Y.H. J. Biol. Chem. 1993; 268: 23981-23985Abstract Full Text PDF PubMed Google Scholar). The conjugation products are more water-soluble, usually less toxic, and can be degraded or transported outside of the cell. Furthermore, GSTs have been implicated in the development of resistance of various tumor cell lines to anti-cancer drugs. A higher level of expression of Alpha class GSTs was observed when Chinese hamster ovary cells were exposed to the nitrogen mustard alkylating agent chlorambucil (7.Lewis A.D. Hickson I.D. Robson C.N. Harris A.L. Hayes J.D. Griffiths S.A. Manson M.M. Hall A.E. Moss J.E. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8511-8515Crossref PubMed Scopus (153) Google Scholar), and cell lines exhibiting overexpression of these GSTs had increased resistance to the anti-cancer drugs chlorambucil and melphalan (8.Puchalski R.B. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2443-2447Crossref PubMed Scopus (164) Google Scholar, 9.Morrow C.S. Smithernan P.K. Diah S.K. Schneider E. Townsend A.J. J. Biol. Chem. 1998; 273: 20114-20120Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar); for recent reviews, see Hayes and Pulford (10.Hayes J.D. Pulford D.J. CRC Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3227) Google Scholar) and Van der Aar et al. (11.Van der Aar E.M. Tan K.T. Commandeur J.N.M. Vermeulen N.P.E. Drug Metab. Rev. 1998; 30: 569-643Crossref PubMed Scopus (11) Google Scholar).The mammalian cytosolic GSTs can be grouped into at least seven classes (Alpha, Mu, Pi, Theta, Kappa, Sigma, and Zeta) (1.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (954) Google Scholar, 12.Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (263) Google Scholar, 13.Board P.G. Baker R.T. Chelvanayagam G. Jermiin L.S. Biochem. J. 1997; 328: 929-935Crossref PubMed Scopus (478) Google Scholar), and they exist as either homo- or heterodimers. Crystal structures have been reported for the representatives of most of these classes (14.Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. Armstrong R.N. Ji X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (387) Google Scholar, 15.Ji X. Zhang P. Armstrong R.N. Gilliland G.L. Biochemistry. 1992; 31: 10169-10184Crossref PubMed Scopus (376) Google Scholar, 16.Reinemer P. Dirr H.W. Ladenstein R. Schaeffer J. Gallay O. Huber R. EMBO J. 1991; 10: 1997-2005Crossref PubMed Scopus (330) Google Scholar, 17.Ji X. Tordova M. O'Donnell R. Parsons J.F. Hayden J.B. Gilliland G.L. Zimniak P. Biochemistry. 1997; 36: 9690-9702Crossref PubMed Scopus (91) Google Scholar, 18.Oakley A.J. Rossjohn J. Lo Bello M. Caccuri A.M. Federici G. Parker M.W. Biochemistry. 1997; 36: 576-585Crossref PubMed Scopus (117) Google Scholar, 19.Wilce M.C.J. Board P.G. Feil S.C. Parker M.W. EMBO J. 1995; 14: 2133-2143Crossref PubMed Scopus (217) Google Scholar, 20.Ji X. Von Rosenvinge E.C. Johnson W.W. Tomarev S.I. Piatigorsky J. Armstrong R.N. Gilliland G.L. Biochemistry. 1995; 34: 5317-5328Crossref PubMed Scopus (215) Google Scholar). All structures share similar topology, with each enzyme subunit having a glutathione binding site and an electrophilic substrate binding site. In addition, a nonsubstrate steroid binding site for Alpha class glutathione S-transferase, isoenzyme 1-1, was located in the cleft formed between the two enzyme subunits (21.McTigue M.A. Williams D.R. Tainer J.A. J. Mol. Biol. 1995; 246: 21-27Crossref PubMed Scopus (283) Google Scholar, 22.Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar). The two subunits in the structures of isoenzymes complexed with various products or product analogues show no significant structural difference between them, suggesting that the two active sites in the enzyme dimer act independently (1.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (954) Google Scholar). Results from steady state kinetics using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene as electrophilic substrates for rat Alpha class enzyme GST 1-1 2Glutathione S-transferase, isoenzyme 1-1, is designated as the rGST A1,2–1,2 isoenzyme in the proposed nomenclature by Hayes and Pulford (10.Hayes J.D. Pulford D.J. CRC Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3227) Google Scholar). GlutathioneS-transferase, isoenzyme 2-2, is designated as the rGST A3–3 isoenzyme. 2Glutathione S-transferase, isoenzyme 1-1, is designated as the rGST A1,2–1,2 isoenzyme in the proposed nomenclature by Hayes and Pulford (10.Hayes J.D. Pulford D.J. CRC Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3227) Google Scholar). GlutathioneS-transferase, isoenzyme 2-2, is designated as the rGST A3–3 isoenzyme. and μ class enzymes GST 3–3 and GST 3–4 are consistent with two noncooperative active sites (23.Ivanetich K.M. Goold R.D. Sikakana C.N. Biochem. Pharmacol. 1990; 39: 1999-2004Crossref PubMed Scopus (33) Google Scholar). However, the large bulky aflatoxin-glutathione conjugate has been shown recently to bind to mouse Alpha class 2-2 enzyme with a stoichiometry of 1 mol/mol of enzyme dimer, and binding of this ligand completely abolished the catalytic activity of both enzyme subunits (24.McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In addition, binding studies of glutathione to the human Pi class enzyme show that binding displays positive cooperativity above 35 °C, whereas negative cooperativity occurs below 25 °C (25.Caccuri A.M. Antonini G. Ascenzi P. Nicotra M. Nuccetelli M. Mazzetti A.P. Federici G. Lo Bello M. Ricci G. J. Biol. Chem. 1999; 274: 19276-19280Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). These results suggest that the two active sites might not be independent. In this paper, we use the bulky product analogue glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP) as a photoaffinity label to probe the interactions between the two enzyme subunits.GS-Succ-BP was used previously in this laboratory as a photoaffinity label for the electrophilic substrate site of the rat liver Mu class GST 4–4 (26.Wang J. Bauman S. Colman R.F. Biochemistry. 1998; 37: 15671-15679Crossref PubMed Scopus (23) Google Scholar). In that study, Met-112 was identified as the reaction site. The benzophenone moiety of GS-Succ-BP binds in the cleft between the subunits, and modification of Met-112 of one enzyme subunit not only prevents the binding and the modification of the corresponding methionine residue on the other subunit but also inhibits the enzyme activities of both enzyme subunits. Comparison between the sequences of rat subunits 4 and 1 reveals only 29% identical plus similar amino acid residues. The sequence differences are undoubtedly responsible for the marked distinctions in substrate specificities between the 4–4 and 1-1 enzymes. In this paper, we show that GS-Succ-BP reacts with the Met-208 of the rat liver Alpha class GST 1-1. This residue, unlike Met-112 of GST 4–4, is located away from the enzyme dimer interface. Modification of Met-208 of one enzyme subunit completely abolishes the catalytic activities of the enzyme dimer. Examination of the catalytic activities of the heterodimer GST 1-2, which was generated from the modified GST 1-1 and GST 2-2, suggests that two enzyme active sites of the dimer are coordinated in the homodimer GST 1-1 but not in the heterodimer GST 1-2.DISCUSSIONGS-Succ-BP functions as a photoaffinity label for rat liver GST 1-1. The initial rate of inactivation shows a nonlinear dependence on the concentration of the reagent, suggesting that a reversible enzyme-reagent complex forms prior to the irreversible modification of the enzyme. Protection against inactivation by the reagent is afforded by the long chain glutathione analogues (S-hexylglutathione or p-nitrobenzylglutathione) or the electrophilic substrate analogues (ethacrynic acid or dinitrobenzene), but not by the short chain glutathione analogue, S-methylglutathione. These results indicate that the reagent binds to the enzyme and reacts within its electrophilic substrate binding site. Steroids (Δ5-androstene-3,17-dione and 17β-estradiol-3,17-disulfate) afford little protection against inactivation, suggesting that the reagent binds and reacts outside of the steroid binding sites of the enzyme.Inactivation of the enzyme is proportional to the covalent incorporation of the reagent into the enzyme. When the enzyme is completely inactivated, an incorporation of about 0.5 mol of reagent per mol of enzyme subunit is observed. Met-208 was identified as the only amino acid being modified by GS-Succ-BP. The location of this residue is quite different from Met-112 of GST 4–4, which was the reaction site of GS-Succ-BP (26.Wang J. Bauman S. Colman R.F. Biochemistry. 1998; 37: 15671-15679Crossref PubMed Scopus (23) Google Scholar). (Met-112 is not conserved in GST 1-1.) Met-208 is located at the beginning of the C-terminal region of GST 1-1, which undergoes a structural change upon binding substrate analogues (14.Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. Armstrong R.N. Ji X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (387) Google Scholar, 44.Schramm V.L. McCluskey R. Emig F.A. Litwack G. J. Biol. Chem. 1984; 259: 714-722Abstract Full Text PDF PubMed Google Scholar, 46.Nieslanik B.S. Dabrowski M.J. Lyon R.P. Atkins W.M. Biochemistry. 1999; 38: 6971-6980Crossref PubMed Scopus (35) Google Scholar).The docking of GS-Succ-BP into the enzyme was modeled, as described under “Experimental Procedures.” The structure of the rat GST 1-1 from residue 1 to 208 was based on the x-ray coordinates of the human GST 1-1 apoenzyme (1GSD in the PDB). The glutathionyl moiety of the reagent was positioned in one of the enzyme's active sites so that it coincided with the glutathionyl of S-benzylglutathione bound to the human GST A1-1 (1GUH in the PDB). Bonds between the sulfur atom of the glutathione and succinimidylbenzophenone, between the sulfur atom and the rest of the glutathione moiety, and between the nitrogen atom of the succinimidyl and benzophenone were rotated, while the intermolecular van der Waals potential and electrostatic potential between the apoenzyme and the reagent was monitored in order to position the carbonyl group of the benzophenone moiety close enough to react with the methyl group of Met-208 without significant steric hindrance from other enzymatic groups. Fig.9 shows the model that resulted from the overall energy minimization of the initial model of the enzyme-reagent complex (with the reagent in green and yellow). In this structure, the Met-208 residue (shown in red) is pointed away from the cleft of the enzyme dimer interface, which was previously identified as the location of the nonsubstrate steroid binding sites of the enzyme (21.McTigue M.A. Williams D.R. Tainer J.A. J. Mol. Biol. 1995; 246: 21-27Crossref PubMed Scopus (283) Google Scholar, 22.Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar). In contrast, our earlier results from the photoaffinity labeling of GST 4–4 with GS-Succ-BP led to the conclusion that the benzophenone moiety of the reagent actually binds in the cleft between the two enzyme subunits. The results indicate that the large reagent (and corresponding large electrophilic substrates) binds distinctively in different isoenzymes. The electrophilic substrate does not protrude into the steroid binding site of the enzyme in the Alpha class isoenzyme, as suggested by McHugh et al. (24.McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar).The C terminus (starting from Met-208) of the human GST 1-1 is not observed in the crystal structure of the apoenzyme (presumably, it is mobile), while it forms a helical cap over the active site of the enzyme enclosing the electrophilic substrate in the structure of enzyme complexed with EA-glutathione or with S-benzylglutathione (14.Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. Armstrong R.N. Ji X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (387) Google Scholar, 45.Cameron A. Sinning I. L'Hermite G. Olin B. Board P. Mannervik B. Jones A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The C-terminal helix in the enzyme-S-benzylglutathione complex (1GUH) is shown inwhite in Fig. 9. Severe steric hindrance exists between the reagent modeled into the apoenzyme and residues of this helix, suggesting that in the enzyme-GS-Succ-BP complex, the position of this helix is not the same as that of enzyme S-benzylglutathione complex; probably, it is a little bit lower to accommodate the bulky moiety of our reagent.The dissociation rate of GS-Succ-BP from the enzyme-reagent complex is extremely slow. This observation is probably due to the dynamics of the C-terminal structural transition between enzymes with and without the large product analogue. It has been shown that the binding of the product analogue and the enzyme involves a conformational change (isomerization) of the enzyme-reagent complex, which mainly involves the helix formation of the C terminus of the enzyme (46.Nieslanik B.S. Dabrowski M.J. Lyon R.P. Atkins W.M. Biochemistry. 1999; 38: 6971-6980Crossref PubMed Scopus (35) Google Scholar). For the large electrophilic substrates of this enzyme, the low catalytic activity is probably due to the slow rate of the product release from the enzyme.In this study, when the enzyme is completely inactivated, only one of the enzyme subunits of the enzyme dimer is modified. This result is consistent with our observation that only 1 mol of reagent binds noncovalently with 1 mol of enzyme dimer in the dark. This finding is somewhat surprising, because each subunit of the enzyme dimer has an active site, and, in the crystal structure of the human GST 1-1 complexed with the EA-glutathione conjugate (1GSE in PDB) or in the crystal structure of GST 1-1 complexed withS-benzylglutathione (1GUH in PDB), it is clear that both active sites of the enzyme are occupied by the product (or product analogue). It has been reported that larger conjugation products (or product analogues) bind to Alpha class enzymes with a stoichiometry of 1 mol/mol of enzyme dimer:S-[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathione for GST 1-1 (44.Schramm V.L. McCluskey R. Emig F.A. Litwack G. J. Biol. Chem. 1984; 259: 714-722Abstract Full Text PDF PubMed Google Scholar) and 8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1 for mouse mGSTA3–3 (24.McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), respectively, while the smaller product analogue p-nitrobenzylglutathione binds to the enzyme with a stoichiometry of 2 mol/mol of mouse GST 2-2 enzyme dimer (24.McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The structures of these glutathione conjugates are shown in Fig. 10. The reagent we are using, GS-Succ-BP, is much larger than p-nitrobenzylglutathione,S-2,4-dinitrobenzylglutathione, and EA-glutathione conjugate but smaller than 8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1. Our results indicate that the binding of GS-Succ-BP to the enzyme is similar to that of the larger glutathione conjugate.Figure 10Selected product analogues of glutathioneS-transferase. AFB-GSH, 8,9-Dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1; SL-GSH,S-[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathione;S-DNP-GSH,S-(2,4-dinitrobenzyl)glutathione;p-nitrobenzyl-GSH,S-(p-nitrobenzyl)glutathione; EA-GSH, glutathionyl-ethacrynic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The modification of one subunit of the enzyme dimer abolishes the enzyme activities on both enzyme subunits. This result is consistent with the observation that the binding of one large conjugation product to one enzyme dimer completely inhibits the enzyme catalytic activity toward CDNB (24.McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In our study, the unmodified subunit of the GS-Succ-BP-modified enzyme can still bind the electrophilic substrate monobromobimane, which then reacts with both Cys residues in the electrophilic substrate binding site of the unmodified subunit. This result suggests that loss of the enzyme activity of the unmodified subunit is probably not due either to the physical barrier to the substrates binding or to the lower binding affinity for substrates induced by the modification of the other subunit but rather results from a conformational change in the unlabeled subunit induced by the modification of the other subunit. This conformational change may cause the substrates to bind in a nonproductive way in the unmodified enzyme. The secondary structures (monitored by CD spectra) show no significant difference between the control enzyme and the modified enzyme, indicating that this conformational change is subtle.It has been generally accepted that the enzyme active sites in the two subunits of glutathione S-transferases are independent (1.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (954) Google Scholar), although it has been shown that the binding of the glutathione to human Pi class enzyme is negatively cooperative at low temperatures and positively cooperative at high temperatures (25.Caccuri A.M. Antonini G. Ascenzi P. Nicotra M. Nuccetelli M. Mazzetti A.P. Federici G. Lo Bello M. Ricci G. J. Biol. Chem. 1999; 274: 19276-19280Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In this study, we observe that the modification of one enzyme subunit of the GST 1-1 homodimer not only abolishes the enzyme catalytic activity of that subunit but also inhibits the catalytic activity of the other subunit of the dimer. This finding suggests that the two enzyme active sites in GST 1-1 are coordinated. On the contrary, results of activity measurements from the GST 1-2 generated from the modified GST 1-1 and control GST 2-2 indicate that modification of the GST 1 subunit of the heterodimer has no effect on the catalytic activity of the GST 2 subunit. Thus, the two active sites in the heterodimer GST 1-2 appear to be independent. These two isoenzymes share 68% sequence identity. It remains to be seen what interaction(s) between the two enzyme subunits cause the active sites to be coordinated in the case of homodimers (GST 1-1) but independent in the case of heterodimers (GST 1-2). Additional study is needed to ascertain whether our results from this pair of homo- and heterodimers are specific or can be generalized to all isoenzymes.In recent years, equilibrium unfolding studies have been performed on Pi class (47.Dirr H.W. Reinemer P. Biochem. Biophys. Res. Commun. 1991; 180: 294-300Crossref PubMed Scopus (54) Google Scholar, 48.Aceto A. Caccuri A.M. Sacchetta P. Bucciarelli T. Dragani B. Rosato N. Federici G. Di Ilio C. Biochem. J. 1992; 285: 241-245Crossref PubMed Scopus (68) Google Scholar, 49.Erhardt J. Dirr H.W. Eur. J. Biochem. 1995; 230: 614-620Crossref PubMed Scopus (53) Google Scholar), Schistosoma japonicum (50.Kaplan W. Husler P. Klump H. Erhardt J. Sluis-Cremer N. Dirr H.W. Protein Sci. 1997; 6: 299-306Crossref Scopus (125) Google Scholar), Alpha class (51.Wallace L.A. Sluis-Cremer N. Dirr H.W. Biochemistry. 1998; 37: 5320-5328Crossref PubMed Scopus (72) Google Scholar), and Sigma class (52.Stevens J.M. Hornby J.A.T. Armstrong R.N. Dirr H.W. Biochemistry. 1998; 37: 15534-15541Crossref PubMed Scopus (38) Google Scholar) glutathione S-transferases. In these studies, urea or guanidinium chloride was added to the enzyme solution to cause denaturation and inactivation, and its dye binding ability, as well as UV, circular dichroism, and fluorescence spectral characteristics were monitored to study the unfolding pathway. GSTs from Pi class, Schistosoma japonicum and Alpha class unfold via a two-state pathway in which only folded dimers or unfolded monomers are detectable at equilibrium (47.Dirr H.W. Reinemer P. Biochem. Biophys. Res. Commun. 1991; 180: 294-300Crossref PubMed Scopus (54) Google Scholar, 48.Aceto A. Caccuri A.M. Sacchetta P. Bucciarelli T. Dragani B. Rosato N. Federici G. Di Ilio C. Biochem. J. 1992; 285: 241-245Crossref PubMed Scopus (68) Google Scholar, 49.Erhardt J. Dirr H.W. Eur. J. Biochem. 1995; 230: 614-620Crossref PubMed Scopus (53) Google Scholar, 50.Kaplan W. Husler P. Klump H. Erhardt J. Sluis-Cremer N. Dirr H.W. Protein Sci. 1997; 6: 299-306Crossref Scopus (125) Google Scholar, 51.Wallace L.A. Sluis-Cremer N. Dirr H.W. Biochemistry. 1998; 37: 5320-5328Crossref PubMed Scopus (72) Google Scholar), while Sigma class GST unfolds via a partially active dimeric intermediate and an inactive monomeric intermediate (52.Stevens J.M. Hornby J.A.T. Armstrong R.N. Dirr H.W. Biochemistry. 1998; 37: 15534-15541Crossref PubMed Scopus (38) Google Scholar). In most of these studies, the refolding of the urea or guanidinium chloride-denatured enzymes was accomplished by dilution (at least 10-fold) in buffer without the denaturant. The enzyme catalytic activity recovered varied from 40–55% in the case of Pi enzyme (48.Aceto A. Caccuri A.M. Sacchetta P. Bucciarelli T. Dragani B. Rosato N. Federici G. Di Ilio C. Biochem. J. 1992; 285: 241-245Crossref PubMed Scopus (68) Google Scholar) to 95–98% for the Alpha enzyme (51.Wallace L.A. Sluis-Cremer N. Dirr H.W. Biochemistry. 1998; 37: 5320-5328Crossref PubMed Scopus (72) Google Scholar). In this study, we were able not only to dissociate the enzyme dimer unequivocally but also to reassociate and recover the enzyme dimer without loss of the protein and the enzyme activities. This approach provides a method to directly study the interaction between enzyme subunits and to elucidate the residue(s) that determines the specificity during dimer formation.In summary, GS-Succ-BP acts as a photoaffinity label for rat GST 1-1, modifying the Met-208 residue. Modification of one subunit of the enzyme dimer completely abolishes the enzyme catalytic activity. Analysis of the ligands that protect against photoinactivation and of models of the enzyme structure suggests that the reagent binds outside the steroid binding site of the enzyme, which is in the cleft between the subunits. Furthermore, reaction of GS-Succ-BP with one subunit causes a conformational change that prevents the binding of the reagent to the other subunit and inhibits the enzyme activity on the unmodified subunit. This interaction is absent in GST 1-2. Glutathione S-transferases (GST)1 (EC 2.5.1.18) are a family of detoxification enzymes that catalyze the conjugation reaction of glutathione with a variety of endogenous electrophiles and xenobiotics (1.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (954) Google Scholar, 2.Mannervik B. Danielson U.H. CRC Crit. Rev. Biochem. 1988; 23: 283-337Crossref PubMed Scopus (1669) Google Scholar, 3.Rushmore T.H. Pickett C.B. J. Biol. Chem. 1993; 268: 11475-11478Abstract Full Text PDF PubMed Google Scholar, 4.Wilce M.C.J. Parker M.W. Biochim. Biophys. Acta. 1994; 1205: 1-18Crossref PubMed Scopus (538) Google Scholar). 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