Active-site Residues Governing High Steroid Isomerase Activity in Human Glutathione Transferase A3-3
2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês
10.1074/jbc.m201062200
ISSN1083-351X
AutoresAnn-Sofie Johansson, Bengt Mannervik,
Tópico(s)Pregnancy and Medication Impact
ResumoGlutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Δ5-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 × 105-fold decrease in k cat; the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease ink cat/K m for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutationk cat/K m was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S.K i values for Δ5-androstene-3,17-dione and Δ4-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK a of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK a of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence ofk cat/K m of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK a values, 6.2 and 8.1. The basic limb of the pH dependence of k cat andk cat/K m disappears in the Y9F mutant. Therefore, the higher kinetic pK areflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate. Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Δ5-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 × 105-fold decrease in k cat; the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease ink cat/K m for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutationk cat/K m was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S.K i values for Δ5-androstene-3,17-dione and Δ4-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK a of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK a of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence ofk cat/K m of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK a values, 6.2 and 8.1. The basic limb of the pH dependence of k cat andk cat/K m disappears in the Y9F mutant. Therefore, the higher kinetic pK areflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate. The glutathione transferases (GSTs) 1The abbreviations used are: GSTglutathione transferaseGSHglutathioneADandrostene-3,17-dioneCDNB1-chloro-2,4-dinitrobenzeneH-sitehydrophobic-substrate binding siteo-CF3-CDNB2-chloro-3,5-dinitro-1,1,1-(trifluoromethyl)benzenePEITCphenethylisothiocyanate are generally considered to be part of the cellular defense against electrophiles and to catalyze a variety of conjugations of the tripeptide glutathione (GSH) to electrophilic centra. In general, the reactions render the electrophiles less reactive and the conjugates can be further metabolized and eventually excreted. In addition to their role as detoxication enzymes, the GSTs have been suggested to be involved in different facets of biological signaling. For example, GSTs have been implicated in the synthesis of various prostaglandins (1.Burgess J.R. Chow N.-W. I. Reddy C.C. Tu C.-P. D. Biochem. Biophys. Res. Commun. 1989; 158: 497-502Crossref PubMed Scopus (22) Google Scholar, 2.Beuckmann C.T. Fujimori K. Urade Y. Hayaishi O. Neurochem. Res. 2000; 25: 733-738Crossref PubMed Scopus (80) Google Scholar, 3.Kanaoka Y. Ago H. Inagaki E. Nanayama T. Miyano M. Kikuno R. Fujii Y. Eguchi N. Toh H. Urade Y. Hayaishi O. Cell. 1997; 90: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), in the 5-lipoxygenase pathway (4.Zhao T.J. Singhal S.S. Piper J.T. Cheng J.Z. Pandya U. Clark-Wronski J. Awasthi S. Awasthi Y.C. Arch. Biochem. Biophys. 1999; 367: 216-224Crossref PubMed Scopus (108) Google Scholar), and in interactions with protein kinases of signal transduction systems (5.Adler V. Yin Z.M. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (962) Google Scholar). GST A3-3 is the most recent example of GSTs with a connection to biological signaling (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). GST A3-3 efficiently catalyzes double-bond isomerizations of Δ5-androstene-3,17-dione (Δ5-AD) and of Δ5-pregnene-3,20-dione, intermediates in the biosynthesis of the steroid hormones progesterone and testosterone (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It is noteworthy that this enzyme is expressed selectively in gonads, placenta, and the adrenal gland. These findings indicate that GST A3-3 plays a role in the production of steroid hormones. glutathione transferase glutathione androstene-3,17-dione 1-chloro-2,4-dinitrobenzene hydrophobic-substrate binding site 2-chloro-3,5-dinitro-1,1,1-(trifluoromethyl)benzene phenethylisothiocyanate The double-bond isomerization of Δ5-AD appears to represent an entirely different biological function than the cellular disposition of toxic agents normally associated with GSTs (7.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (963) Google Scholar). However, there are other isomerization reactions catalyzed by GSTs. Thecis-trans conversion of retinoic acid is a GSH-independent reaction catalyzed by GST P1-1 (8.Chen H. Juchau M.R. Biochem. J. 1998; 336: 223-226Crossref PubMed Scopus (23) Google Scholar). Zeta class GSTs are involved in the catabolic pathway of tyrosine and phenylalanine by catalyzing the GSH-dependent cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate (9.Blackburn A.C. Woollatt E. Sutherland G.R. Board P.G. Cytogenet. Cell Genet. 1998; 83: 109-114Crossref PubMed Scopus (73) Google Scholar, 10.Fernández-Cañón J. Peñalva M.A. J. Biol. Chem. 1998; 273: 329-337Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Rat GST A1-1 and GSTs in human liver cytosol have been shown to catalyze the tautomerization of 2-hydroxymenthofuran, a reaction that strictly requires GSH and the phenolic hydroxyl group of Tyr-9 (11.Khojasteh-Bakht S.C. Nelson S.D. Atkins W.M. Arch. Biochem. Biophys. 1999; 370: 59-65Crossref PubMed Scopus (18) Google Scholar). In the latter case a general base-catalyzed isomerization was proposed, in which the thiolate form of GSH serves to deprotonate the substrate and initiate the reaction. This mechanism is similar to the double-bond shift in 3-oxo steroids catalyzed by GST A1-1 (12.Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and GST A3-3 (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). However, in the isomerizations catalyzed by Zeta class GSTs, GSH is reversibly added as a nucleophile to the substrate rather than functioning as a base, thus representing a mechanism more closely related to GSH conjugations. The members of the Alpha class of GSTs, A1-1, A2-2, and A3-3 are close relatives. From an evolutionary perspective GST A3-3 appears to have diverged from GST A2-2 and GST A1-1, which are the nearest neighbors on the phylogenetic tree (13.Board P.G. Biochem. J. 1998; 330: 827-831Crossref PubMed Scopus (56) Google Scholar). The latter enzymes have been found to function as efficient glutathione-dependent peroxidases (4.Zhao T.J. Singhal S.S. Piper J.T. Cheng J.Z. Pandya U. Clark-Wronski J. Awasthi S. Awasthi Y.C. Arch. Biochem. Biophys. 1999; 367: 216-224Crossref PubMed Scopus (108) Google Scholar). From the evolutionary branch point GST A3-3 has accumulated mutations in the active site that make it an efficient steroid double-bond isomerase. Despite the high sequence identity (88%) between GST A3-3 and GST A2-2, they differ considerably in their isomerase activities with the steroid Δ5-AD, GST A3-3 displaying a 5000-fold higher catalytic efficiency (k cat/K m). Five of the 26 residues (out of 222, including the initiator methionine) that differ between GST A2-2 and GST A3-3 are located in the hydrophobic substrate-binding site (H-site). In this study, we have investigated the determinants for the high steroid isomerase activity of GST A3-3, and we propose the outline of a reaction mechanism for the isomerization reaction. 1-Chloro-2,4-dinitrobenzene (CDNB), reduced glutathione (GSH), and 2-chloro-3,5-dinitro-1,1,1-(trifluoromethyl)benzene (o-CF3-CDNB) were purchased from Sigma Chemical Co. (St. Louis, MO). Δ4-Androstene-3,17-dione and phenethylisothiocyanate were purchased from Aldrich (Milwaukee, WI). Δ5-Androstene-3,17-dione was obtained from Steraloids Inc. (Newport, RI). All oligonucleotides were purchased from Interactiva (Ulm, Germany). Restriction enzymes were from Roche Diagnostics (Mannheim, Germany), and Pfu DNA polymerase was obtained from Stratagene (La Jolla, CA). The DNA vectors pGEM-3Z and pET-21a(+) were obtained from Promega (Madison, WI) and Novagen (Madison, WI), respectively. Escherichia coli XL1-Blue was purchased from Stratagene and E. coli BL-21(DE3) from Novagen. The cDNA of GSTA3 was amplified from the original expression clone pKK-DA3 (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) using PCR with the oligonucleotides A3EcoRINdeIG9T: 5′-ATATGAATTC ATATGGCAGGTAAGCCCAAGCTTCAC-3′ (the EcoRI and NdeI restriction sites are underlined and shown in boldface, respectively), A3NCRev 5′-AATAATGTCGACTTGTTAGCCTGGATGGCTGCT-3′ (theSalI site is underlined). The primer A3EcoRINdeIG9T introduces a silent G to T mutation at nucleotide position 9 in the cDNA of GSTA3 to replace a codon seldom used in highly expressed genes in E. coli with a more commonly used codon. The PCR product was subcloned into the pGEM vector using EcoRI andSalI. The GSTA3 cDNA was sequenced using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (USB Corporation, Cleveland, OH) to verify that no mutations had been introduced. The GSTA3 cDNA was subsequently subcloned into the expression vector pET-21a(+) giving the expression clone pET-21a(+)GSTA3. Approximately 65 mg of pure GST A3-3 was successfully obtained per liter of culture medium, a 40-fold increase compared with the yield obtained from the expression system previously used (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The GST A3-3 mutants, GST A3-3/F10S, GST A3-3/L111F, GST A3-3/A216S, GST A3-3/F10S/A216S, GST A3-3/F10S/L111F/A216S, and GST A3-3/Y9F, were constructed using inverted PCR (14.Sambrook J. Russell D.W. Molecular Cloning. 3rd Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 8.81-8.85Google Scholar) with mutagenic oligonucleotides, clonedPfu DNA polymerase, and the appropriate cDNA in the pGEM-3Z vector as template. The PCR conditions used were 95 °C at 10 min followed by the cycle, 95 °C for 1 min, 54 °C for 1 min and 30 s, and 72 °C for 10 min, repeated 35 times followed by 20 min at 72 °C. The mutant cDNAs were sequenced and subcloned into the expression vector pET-21a(+) using the restriction sitesNdeI and SalI. The proteins were expressed from the pET-21a(+) vector in E. coli BL-21(DE3). The cells were grown to A 600 = 0.7, and expression was induced by addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside. The cells were grown for 4 h, collected by centrifugation, and lysed using sonication. The lysate was desalted on a PD-10 gel filtration column (Amersham Biosciences, Inc.), and the proteins were eluted in 20 mm sodium phosphate, pH 7.0, and were subsequently loaded onto a HiTrap SP cation exchanger (Amersham Biosciences, Inc.). The proteins were eluted using a salt gradient. This single purification step yielded highly pure enzymes as confirmed by SDS-PAGE stained with Coomassie Brilliant Blue. All the mutants were obtained in yields similar to that of the wild-type enzyme. The extinction coefficient 23,900 m−1 cm−1 at 280 nm for the GSTA3 subunit (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) was used to determine the subunit concentration. The specific activities of GST A3-3 and all the constructed GST A3-3 mutants were determined for the isomerization reaction with Δ5-AD (see Fig. 1 A) and for the conjugation reaction with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH (Fig. 1 B). The reactions were monitored spectrophotometrically at 30 °C. The isomerization of 100 μm Δ5-AD was followed at 248 nm in 25 mm sodium phosphate buffer, pH 8.0, in the presence of 1 mm GSH. The extinction coefficient for the product Δ4-AD is 16,300 m−1cm−1 (15.Benson A.M. Talalay P. Keen J.H. Jakoby W.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 158-162Crossref PubMed Scopus (156) Google Scholar). Specific activity measurements with 1 mm CDNB were performed in 0.1 m sodium phosphate, pH 6.5, in the presence of 1 mm GSH as described previously (16.Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). All steady-state kinetic measurements were performed at 30 °C. The isomerization activity of GST A3-3 and the GST A3-3 mutants was monitored at pH 8.0, in 25 mm sodium phosphate at a close-to-saturating concentration of GSH (2 mm). The concentration of Δ5-AD was varied between 1 and 200 μm. In addition, saturation curves with Δ5-AD were measured in the absence of GSH for GST A3-3 and GST A3-3/Y9F. The conjugating activities of GST A3-3, GST A3-3/L111F, GST A3-3/F10S/L111F/A216S, and GST A3-3/Y9F with CDNB were measured in 0.1 m sodium phosphate pH 6.5 at 5 mm GSH and with phenethylisothiocyanate (PEITC) (Fig. 1 C) in 0.1 msodium phosphate, pH 7.4, at 5 mm GSH as described (17.Meyer D.J. Crease D.J. Ketterer B. Biochem. J. 1995; 306: 565-569Crossref PubMed Scopus (97) Google Scholar). The concentrations of CDNB ranged between 25 and 1500 μm, and the concentration of phenethylisothiocyanate was varied between 5 and 400 μm. Kinetic parameters were determined from the data using non-linear regression analysis. Catalytic center activities (k cat) were expressed per subunit (25,300 Da). The ionization of Tyr-9 was measured as a function of pH by UV absorption difference spectroscopy (18.Björnestedt R. Stenberg G. Widersten M. Board P.G. Sinning I. Jones T.A. Mannervik B. J. Mol. Biol. 1995; 247: 765-773Crossref PubMed Scopus (102) Google Scholar). The intrinsic absorbance at 253 nm of 10–15 μmsubunits of the different enzyme variants was measured in 0.1m sodium phosphate buffer at pH 5.5–8.0 and 0.1m ethanolamine/HCl at pH 8.2–9.0 at 22 °C. The spectrum of the same protein sample at pH 5.5 was subtracted from the spectra obtained at other pH values. The equation ΔA = ΔAmax/[1 + 10( p Ka− pH)] was fitted to the data using rffit provided in the SIMFIT package (19.Bardsley W.G. Bukhari N.A.J. Ferguson M.W.J. Cachaza J.A. Burguillo F.J. Comput. Chem. 1995; 19: 75-84Crossref Scopus (48) Google Scholar). The effect of adding a saturating concentration of Δ4-AD (100 μm) or GSH (2 mm) on the pK a of Tyr-9 of wild-type GST A3-3 was monitored by measuring the intrinsic absorbance at 300 nm, because both molecules absorb strongly at 253 nm. To calculate the number of tyrosinates per subunit, the ΔA 253 and ΔA 300 values were divided by the subunit concentration used and the extinction coefficient determined for GST tyrosines at 253 and 300 nm, 11,000 and 2350m−1 cm−1, respectively (20.Atkins W.M. Dietze E.C. Ibarra C. Protein Sci. 1997; 6: 873-881Crossref PubMed Scopus (27) Google Scholar,21.Dietze E.C. Ibarra C. Dabrowski M.J. Bird A. Atkins W.M. Biochemistry. 1996; 35: 11938-11944Crossref PubMed Scopus (29) Google Scholar). The pK a of GSH bound to the active site of GST A3-3 and GST A3-3/F10S/L111F/A216S was determined from spectra of 10 μm subunits in the presence of 0.5 mm GSH in the sample cuvette. Difference spectra were obtained by subtracting the spectrum for the enzyme alone at pH 5.5 and the spectra of GSH at the corresponding pH values from the spectrum of the enzyme·GSH complex. The peak at 239 nm arising from the thiolate was plotted against pH and the equation ΔA = ΔAmax/[1 + 10( p Ka− pH)] was fitted to the data points using rffit (19.Bardsley W.G. Bukhari N.A.J. Ferguson M.W.J. Cachaza J.A. Burguillo F.J. Comput. Chem. 1995; 19: 75-84Crossref Scopus (48) Google Scholar). To calculate the number of ionized GSH molecules per protein subunit the extinction coefficient 5200m−1 cm−1 at 239 nm determined for the thiolate anion in the GST active site was used (22.Graminski G.F. Kubo Y. Armstrong R.N. Biochemistry. 1989; 28: 3562-3568Crossref PubMed Scopus (163) Google Scholar). The isomerase activity of GST A3-3, GST A3-3/Y9F, and GST A3-3/F10S/L111F/A216S with Δ5-AD in the presence of 2 mm GSH was measured at pH intervals of ∼0.4 pH units in the pH range 5.7–8.8. Below pH 8.0 the measurements were performed in 0.1 m sodium phosphate, and 0.1 methanolamine/HCl was used above pH 8.0. The concentration of Δ5-AD was varied between 1 and 200 μm. The affinity between the steroids Δ5-AD or Δ4-AD and wild-type GST A3-3, GST A3-3/L111F, and GST A3-3/F10S/L111F/A216S was studied by inhibition experiments. When Δ4-AD was used as an inhibitor, the concentration of the substrate Δ5-AD was varied between 2.5 and 400 μm in 25 mm sodium phosphate at pH 8.0. The activity was measured in the absence and presence of 400 μm Δ4-AD. The inhibitory potency of Δ5-AD was determined by means of competition experiments with the alternative substrate o-CF3-CDNB using 100 μm Δ5-AD in 0.1 m sodium phosphate, pH 6.5, at a fixed GSH concentration of 2 mm. The concentration of o-CF3-CDNB was varied between 25 and 700 μm. The initial velocities were determined spectrophotometrically at 30 °C, and the equation describing competitive inhibition was fitted to the data by non-linear regression analysis. Sequence alignment of the Alpha class GSTs and structural information available for the close relative GST A1-1 (23.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 (415) Google Scholar) were used to identify residues in GST A3-3 that are potential determinants for the high steroid isomerase activity. GST A2-2, which displays a lower steroid double-bond isomerase activity by three orders of magnitude as compared with GST A3-3 (6.Johansson A.-S. Mannervik B. J. Biol. Chem. 2001; 276: 33061-33065Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 12.Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), differs from GST A3-3 in 26 amino acids out of 222 (including the initiator methionine). Five of these residues are situated in the H-site as judged from the crystal structure (23.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 (415) Google Scholar). The mutations in GST A3-3 required to mimic the H-site of GST A2-2 are F10S, I12G, L111F, A208M, and A216S. The mutations F10S and A216S change the topography of the active site and introduce polar residues with hydrogen bonding potential. Because Δ5-AD is a highly hydrophobic substrate, one would expect the introduction of polar residues to lower the binding affinity by providing a less hydrophobic environment in the active site. The active site of the highly efficient Δ5-3-ketosteroid isomerase in bacteria is very hydrophobic (24.Wu Z.R. Ebrahimian S. Zawrotny M.E. Thornburg L.D. Perez-Alvarado G.C. Brothers P. Pollack R.M. Summers M.F. Science. 1997; 276: 415-418Crossref PubMed Scopus (140) Google Scholar, 25.Kim S.W. Cha S.S. Cho H.S. Kim J.S. Ha N.C. Cho M.J. Joo S. Kim K.K. Choi K.Y. Oh B.H. Biochemistry. 1997; 36: 14030-14036Crossref PubMed Scopus (132) Google Scholar) suggesting that introduction of polar residues in the active site of GST A3-3 would impair the isomerase activity. Hydrogen bonding between the serine residues, and the keto groups of the steroid may also lead to non-productive binding of the substrate. A crystal structure of GST A3-3 is not yet solved nor is any other relevant structure available for a GST in complex with a steroid such as Δ4-AD. However, in all structures of GST A1-1 crystallized with a hydrophobic S-substituent on GSH in the active site, residue 111 is identified as a residue that lines the H-site with its side chain oriented toward the hydrophobic moiety (23.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 (415) Google Scholar,26.Cameron A.D. Sinning I. L'Hermite G. Olin B. Board P.G. Mannervik B. Jones T.A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). An increase of the volume of residue 111 might affect productive binding of a large substrate through steric hindrance. To study the role of residues 10, 111, and 216 in the enzymatic function of GST A3-3, the separate and combined mutations F10S, L111F, and A216S were constructed, introducing the amino acids of GST A2-2 in the positions of the variant residues. In other GSTs Tyr-9 has been shown to be a catalytically important residue (27.Stenberg G. Board P.G. Mannervik B. FEBS Lett. 1991; 293: 153-155Crossref PubMed Scopus (95) Google Scholar, 28.Kolm R.H. Sroga G.E. Mannervik B. Biochem. J. 1992; 285: 537-540Crossref PubMed Scopus (87) Google Scholar). The Y9F mutant was constructed to investigate the function of the phenolic hydroxyl group of Tyr-9 also in GST A3-3. The ionization of tyrosine residues in GST A3-3, GST A3-3/L111F, GSTA3-3/F10S/L111F/A216S, and GST A3-3/Y9F was monitored as a function of pH by measuring the intrinsic absorbance of the enzyme at 253 nm. The titration curves are shown in Fig. 2. Ionization of tyrosines other than Tyr-9 in the enzyme makes only minor contributions in the pH range used as shown by titration of GST A3-3/Y9F (Fig. 2). The L111F mutation in GST A3-3 increases the pK a value of Tyr-9 from 7.93 ± 0.04 to 8.25 ± 0.08. Addition of the point mutations F10S and A216S affords an additional shift of 1 pH unit to a pK a value of 9.2, ∼1.3 pH units higher than that of GST A3-3 and approaching the pK a value of tyrosine in aqueous solution, 10.1 (29.Creighton T.E. Proteins: Structures and Molecular Properties. 2nd Ed. W. H. Freeman & Co., New York1993: 16Google Scholar). The effect of adding a saturating concentration of Δ4-AD (100 μm) or GSH (2 mm) on the pK a of Tyr-9 of wild-type GST A3-3 was monitored by measuring the tyrosinate absorbance at 300 nm. This higher wavelength was chosen, because both ligands absorb strongly at 253 nm. Δ4-AD did not affect the pK a value of Tyr-9 (data not shown), but the presence of 2 mm GSH increased the pK avalue of Tyr-9 by ∼1 pH unit to 8.8 ± 0.2, showing that the ionization of Tyr-9 is linked to the ionization of GSH. The pK a value of GSH bound to the active site of GST A3-3 was determined as 6.1 ± 0.1. This pK avalue was shifted to 7.1 ± 0.2 in the GST A3-3/S10F/L111F/A216S mutant. In the GST A3-3/Y9F mutant the pK a value was increased to 6.5 ± 0.2, demonstrating that the phenolic hydroxyl group of Tyr-9 has only a minor influence on the ionization of the thiol group of GSH, which in aqueous solution has a pK a value of 9.2 (30.Jung G. Breitmaier E. Voelter W. Eur. J. Biochem. 1972; 24: 438-445Crossref PubMed Scopus (118) Google Scholar). The role of Tyr-9, or equivalent residues, in GSTs is generally considered to be stabilization of the thiolate of GSH in the active site by serving as hydrogen bond donor (7.Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (963) Google Scholar). In GST A1-1 the corresponding mutation afforded a similar minor increase in the pK a value of GSH from 6.7 to 7.2 (12.Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This change of 0.5 pH unit corresponds to a 2.8 kJ/mol stabilization of the thiol over the thiolate form. It is notable that this is less than the energy of a fully formed hydrogen bond (31.Fersht A. Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman & Co., New York1999Google Scholar). The pK a value of GSH bound to the active site of GST A3-3/Y9F is 2.7 pH units lower than in solution (30.Jung G. Breitmaier E. Voelter W. Eur. J. Biochem. 1972; 24: 438-445Crossref PubMed Scopus (118) Google Scholar) showing that other groups together contribute more than the phenolic hydroxyl of Tyr-9 to lowering the pK a of GSH. A plausible candidate is Arg-15, which in GST A1-1 lowers the pK a of GSH (18.Björnestedt R. Stenberg G. Widersten M. Board P.G. Sinning I. Jones T.A. Mannervik B. J. Mol. Biol. 1995; 247: 765-773Crossref PubMed Scopus (102) Google Scholar). The pK a of GSH was increased by 1.0 pH unit to 7.1 ± 0.2 in the GSTA3-3/F10S/L111F/A216S mutant. This may be due to an influence of the hydrophilic serines on the first-sphere interactions from Tyr-9 and Arg-15 that contribute to the lowering of the pK a of GSH, in such a way that their thiolate-stabilizing effect is impaired. The specific activities of wild-type GST A3-3 and the constructed GST A3-3 mutants with the substrates Δ5-AD and CDNB are compiled in Table I. For comparison, specific activity values are also given for the homologous GST A1-1, the GST A1-1/Y9F mutant, and GST A2-2. The single mutation that produces the most pronounced difference in specific activity with Δ5-AD is the Y9F mutation, which lowers the specific activity 350-fold. However, this same mutation decreases the specific activity only ∼2-fold with CDNB. In GST A1-1 the corresponding Y9F mutation decreases the specific activity with Δ5-AD and CDNB 43- and 300-fold, respectively (12.Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The hydroxyl group of Tyr-9 in GST A3-3 therefore seems to play a more important role in the catalysis of the double-bond isomerization than in the nucleophilic aromatic substitution reaction of GSH and CDNB. In the case of GST A1-1 Tyr-9 is relatively less important for the isomerase reaction. Also the L111F mutation in GST A3-3 by itself markedly decreases the specific activity with Δ5-AD (27-fold) but affords a notable 4-fold increase in the specific activity with CDNB.Table ISpecific activities of Alpha class GSTs and active-site mutants determined with Δ5-androstene-3,17-dione and 1-chloro-2,4-dinitrobenzeneEnzymeSpecific activity1-aValues are means ± S.E. of at least five measurements.Δ5-ADCDNB(μmolmg−1min−1)GST A3-3285 ± 523 ± 3GST A3-3/A216S290 ± 1022.7 ± 0.3GST A3-3/F10S133 ± 2411.2 ± 0.4GST A3-3/F10S/A216S123 ± 115.7 ± 0.1GST A3-3/L111F10.6 ± 0.285.9 ± 1.5GST A3-3/F10S/L111F/A216S5.1 ± 0.211.8 ± 0.5GST A3-3/Y9F0.8 ± 0.19.2 ± 0.5GST A1-140 ± 31-bValues are from Ref.12.111 ± 81-bValues are from Ref.12.GST A1-1/Y9F0.94 ± 0.121-bValues are from Ref.12.0.37 ± 0.061-bValues are from Ref.12.GST A2-20.172 ± 0.0031-bValues are from Ref.12.58 ± 41-bValues are from Ref.12.1-a Values are means ± S.E. of at least five measurements.1-b Values are from Ref.12. Open table in a new tab Steady-state kinetic parameters are listed in Table II for wild-type and mutated GST A3-3 variants as well as for GST A1-1 and GST A1-1/Y9F catalyzing the isom
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