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Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of Substrate Binding to the Active Site of Glutathione Transferases

2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês

10.1074/jbc.m002819200

1083-351X

Anna Maria Caccuri, Giovanni Antonini, Philip G. Board, Jack U. Flanagan, Michael W. Parker, Roberto Paolesse, Paola Turella, Giorgio Federici, Mario Lo Bello, Giorgio Ricci,

Reproductive Health and Contraception

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK a = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k on andk off values at least hundred times lower (k on = (2.7 ± 0.8) × 104m−1s−1, k off = 36 ± 9 s−1, at 37 °C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK a value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results. Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK a = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k on andk off values at least hundred times lower (k on = (2.7 ± 0.8) × 104m−1s−1, k off = 36 ± 9 s−1, at 37 °C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK a value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results. glutathione transferase 1-menaphthyl sulfate The human cytosolic glutathione transferases (GSTs,1 EC 2.5.1.18) are dimeric proteins grouped into at least four gene independent classes, named Alpha, Mu, Pi, and Theta that differ in amino acid sequence, co-substrate specificity, and antibody cross-reactivity (for reviews, see Refs. 1Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (946) Google Scholar and 2Salinas A.E. Wong M.G. Curr. Med. Chem. 1999; 6: 279-309PubMed Google Scholar). Despite an inter-class sequence identity of less than 25% between Alpha, Mu, and Pi class enzymes and less than 10% to the Theta class GSTs, the three-dimensional fold of these isoenzymes is very similar (3Dirr H.W. Reinemer P. Huber R. Eur. J. Biochem. 1994; 220: 645-661Crossref PubMed Scopus (381) Google Scholar, 4Wilce M.C.J. Parker M.W. Biochim. Biophys. Acta. 1994; 1205: 1-18Crossref PubMed Scopus (537) Google Scholar, 5Wilce M.C.J. Board P.G. Feil S.C. Parker M.W. EMBO J. 1995; 14: 2133-2143Crossref PubMed Scopus (217) Google Scholar, 6Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1997; 6: 309-322Abstract Full Text Full Text PDF Scopus (133) Google Scholar). Notable differences for the human Theta enzyme T2-2 are a small and buried active site for GSH and an extra C-terminal extension of about 40 residues not found in the other classes (6Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1997; 6: 309-322Abstract Full Text Full Text PDF Scopus (133) Google Scholar). Recently a fifth class has been discovered in humans, named Zeta class, showing a serine residue in the active site and high activity toward organic hydroperoxides (7Board P.G. Baker R.T. Chelvanayagam G. Jermiin L.S. Biochem. J. 1997; 328: 929-935Crossref PubMed Scopus (473) Google Scholar). From an evolutionary point of view, it has been proposed that Alpha, Mu, and Pi class GSTs originated from the Theta GST by gene duplication (8Pemble S.E. Taylor J.B. Biochem. J. 1992; 287: 957-963Crossref PubMed Scopus (180) Google Scholar). In turn, on the basis of sequence identity at the N terminus, the Theta GST should might have arisen from the ancestral mitochondrial GST Kappa (9Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (263) Google Scholar). Alternatively, the Theta class may be only older than Alpha, Pi, and Mu GSTs and have all diverged from a common ancestor (7Board P.G. Baker R.T. Chelvanayagam G. Jermiin L.S. Biochem. J. 1997; 328: 929-935Crossref PubMed Scopus (473) Google Scholar). Recently, we have found that human Alpha, Mu, and Pi class GSTs bind GSH by adopting a very similar multistep mechanism in which the final Michaelis complex is achieved after the formation of a weak pre-complex (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar, 11Caccuri A.M. Antonini G. Board P.G. Parker M.W. Nicotra M. Lo Bello M. Federici G. Ricci G. Biochem. J. 1999; 344: 419-425Crossref PubMed Scopus (54) Google Scholar). Does such a mechanism hold for the primitive GSTT2-2? Furthermore, all GSTs activate the substrate by lowering the pK a value of GSH at the active site, but the peculiar sulfatase reaction catalyzed by hGSTT2-2 could not need the thiolate form of GSH (12Tan K.-L. Chelvanayagam G. Parker M.W. Board P.G. Biochem. J. 1996; 319: 315-321Crossref PubMed Scopus (64) Google Scholar). Interestingly, in this old enzyme, Ser-11 replaces the Tyr residue found in Alpha, Pi, and Mu class GSTs. This aromatic residue contacts the sulfhydryl group of GSH and stabilizes its ionized form (1Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (946) Google Scholar). Recently, Jemth and Mannervik (13Jemth P. Mannervik B. Biochemistry. 1999; 38: 9982-9991Crossref PubMed Scopus (21) Google Scholar, 14Jemth P. Mannervik B. J. Biol. Chem. 2000; 275: 8618-8624Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) described some kinetic and binding properties of the rat Theta class GST. They obtained indirect kinetic indications for a forced deprotonation of GSH at the active site, and they also proposed Ser-11 as the crucial residue involved in this activation. We report here, for the human GSTT2-2, direct evidence for the ionization of GSH at the active site, but we point out the crucial role of Arg-107 in the binding and activation of the substrate. This residue contacts the thiol sulfur of GSH, either directly or through a water molecule (6Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1997; 6: 309-322Abstract Full Text Full Text PDF Scopus (133) Google Scholar); replacement of Arg-107 by Ala not only alters the pK a value of the bound GSH but even changes the binding mechanism of this enzyme. Interestingly, some of the binding properties of the old enzyme seem to be related to the rigidity of selected regions of the protein, indicating a possible evolutionary target in terms of flexibility for the GST superfamily. GSH andS-hexylglutathione are Sigma products; sodium 1-menaphthyl sulfate (Msu) was prepared as described by Clapp and Young (15Clapp J.J. Young L. Biochem. J. 1970; 118: 765-771Crossref PubMed Scopus (65) Google Scholar). His-tagged recombinant GST2-2 and R107A mutants were expressed inEscherichia coli and purified using immobilized metal ion chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen) as described previously (12Tan K.-L. Chelvanayagam G. Parker M.W. Board P.G. Biochem. J. 1996; 319: 315-321Crossref PubMed Scopus (64) Google Scholar, 16Flanagan J.U. Rossjohn J. Parker M.W. Board P.G. Chelvanayagam G. Protein Sci. 1999; 8: 2205-2212Crossref PubMed Scopus (13) Google Scholar). The intrinsic fluorescence of hGSTT2-2 was measured in a single photon counting spectrofluorometer (Fluoromax, S.A. Instrument, Paris, France) with a sample holder at 25 °C. Excitation was at 280 nm, and emission was at 335 nm. In a typical experiment, fluorescence intensity was measured before and after the addition of suitable amounts of GSH (from 0.02 to 8 mm) to 1.5μm hGSTT2-2 in 0.1 mpotassium phosphate buffer, pH 7.0. Experimental data were corrected both for dilution and for inner filter effects and fit to Equation 1, FL=Fo+Fmax−Fo/1+[S]0.5/[S]nHEquation 1 where F 0 is the protein fluorescence in the absence of GSH; F L is the protein fluorescence in the presence of a given amount of GSH; F maxis the protein fluorescence at saturating GSH concentrations, andn H is the Hill coefficient. Difference spectra of GSH thiolate bound to both native and the R107A mutant of hGSTT2-2 were obtained with a Kontron double-beam Uvikon 940 spectrophotometer thermostated at 25 °C. In a typical experiment 1 mm GSH was added to the enzyme (15μm active sites) in a suitable buffer. From the resulting spectrum, the contributions from free GSH and free enzyme were subtracted. The amount of thiolate was obtained by assuming an ε240 nm of 5,000m−1 cm−1. The pH dependence of the GSH ionization was obtained with the following buffers (0.01 m): sodium acetate buffer, pH 5.5, and potassium phosphate buffers between pH 6.0 and pH 8.0. pK a values were obtained by fitting the data to Equation 2. y=ylim/1+10pKa−pHEquation 2 Thiol proton extrusion was detected at 25 °C as reported previously (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar). In a typical experiment, performed under a N2 atmosphere, a GSH solution (10 mm in 0.1 m NaCl) was titrated to a fixed pH with 0.1 m NaOH and mixed with the same volume of GSTT2-2 (4 mg/ml in 0.1 m NaCl) at exactly the same pH. Quantitation of the released proton was obtained by suitable addition of 10 mm NaOH up to the initial pH value. A blank was performed at each pH, by replacing GSH withS-hexylglutathione (4 mm in 0.1 mNaCl). pK a values were obtained by fitting the data to Equation 3, y=ylim10−pKa1×10−pH/ 10−pKa1×10−pKa2+10−pKa1×10−pH+10−2pHEquation 3 Rapid kinetic experiments were performed on an Applied Photophysics Kinetic spectrometer stopped-flow instrument equipped with a thermostated 1-cm light path observation chamber. Kinetics of the binding of GSH to wild-type and R107A mutant were studied at 37 °C, by rapid mixing of enzyme (95μm) and different amounts of GSH (from 0.25 to 12 mm) in carbonate/phosphate/acetate (50:50:50μm) buffer, pH 7.0. Binding of GSH was monitored by following the increase of the intrinsic fluorescence of the protein. Experimental traces were fit to a single exponential decay, and pseudo-first order kinetic constants were calculated at different GSH concentrations. Binding of GSH as function of pH was performed at 37 °C. Wild-type or R107A mutant (95μm) in carbonate/phosphate/acetate (50:50:50μm) buffer were rapidly mixed with the same volume of GSH (2.0 mm) dissolved in the same buffer. Experiments were done at different pH values between pH 5.0 and pH 9.0. Binding of GSH was monitored by following the increase of the intrinsic fluorescence of the protein. Experimental traces were fit to a single exponential decay, and pseudo-first order kinetic constants were calculated. Isothermic binding of GSH to the G-site of hGSTT2-2 has been studied at 25 °C by using the perturbation of the intrinsic fluorescence of the protein upon addition of different GSH concentrations. In the native enzyme, the interaction of the substrate with the active site causes an increase of the tryptophan fluorescence, and this is a peculiar finding as other GST isoenzymes display a fluorescence quenching on GSH binding. Binding of GSH is hyperbolic (n H = 1.0) with an [S]0.5 = K d value of 0.8 ± 0.2 mm (Fig. 1 A). This value is at least four times higher than those found for the more recently evolved Alpha, Pi, and Mu class GSTs. This poor affinity for GSH is also reflected by the lack of interaction of this GST with the classical GSH affinity matrix (12Tan K.-L. Chelvanayagam G. Parker M.W. Board P.G. Biochem. J. 1996; 319: 315-321Crossref PubMed Scopus (64) Google Scholar). Direct demonstration of GSH thiolate formation at the active sites of hGSTT2-2 and of R107A mutant and their dependence on pH have been obtained by differential UV spectroscopy. Fig. 1 B, inset, shows a typical thiolate absorption band centered at 240 nm obtained at pH 7.5 by the differential UV spectrum of hGSTT2-2 in the presence of nonsaturating GSH concentration (1 mm). Similar thiolate band is obtained with the R107A mutant. Higher GSH concentrations cannot be used because of the large spectral contribution due to the spontaneous ionization of GSH at alkaline pH values. By assuming an ε240 nm of 5,000m−1 cm−1for the ionized GSH, the limiting value at alkaline pH is 0.48 GS− equivalents per hGSTT2-2 active sites when the active site occupancy is about 55%. The dependence, at pH 7.0, of the GSH thiolate band at 240 nm on GSH concentration (from 0.1 to 1 mm) follows a hyperbolic behavior that overlaps the GSH binding experiments (see Fig. 1 A) (data not shown). It follows that about 0.9 GS−/active sites are formed in the native enzyme at 25 °C under saturating substrate. The pH dependence of the spectral perturbation at 240 nm gives an apparent pK a value for the bound GSH of 6.15 ± 0.10 (Fig. 1 B), close to the pK a values found for Alpha, Mu, and Pi class GSTs (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar, 11Caccuri A.M. Antonini G. Board P.G. Parker M.W. Nicotra M. Lo Bello M. Federici G. Ricci G. Biochem. J. 1999; 344: 419-425Crossref PubMed Scopus (54) Google Scholar). Mutation of Arg-107 remarkably decreases the deprotonation of the substrate, and a pK a = 7.8 ± 0.2 for the thiol group of the bound GSH is now obtained (Fig. 1 B), a value 1.6 pH units higher than that calculated for the native enzyme. The fate of the proton produced from the GSH ionization has been investigated in hGSTT2-2 by a potentiometric approach as described under “Experimental Procedures.” In a typical experiment (pH 6.5), a nearly saturating GSH solution (5 mm final concentration) was mixed with hGSTT2-2 (73μm final concentration), both solutions in the absence of any buffer, and titrated to the same pH value. After mixing, a rapid decrease of pH was observed. No pH changes have been found when the GSH analogue S-hexylglutathione replaces GSH. These experiments show that in hGSTT2-2, protons are released upon GSH binding and that they come from the sulfhydryl group of GSH. Back titration with dilute NaOH allows an estimation of the amount of the released proton. The pH dependence of this event shows a bell-shaped trend (Fig. 2). It is evident that under alkaline conditions at least one protein residue acts as an internal base for proton neutralization. By fixing the pK a1 = 6.15 (the value obtained for GSH ionization by differential UV spectroscopy), the best fit of the experimental data gives an apparent pK a2of 7.32 ± 0.03 for the unknown protein base. Kinetics of GSH binding was studied at pH 7.0 and 37 °C, by following the increase of protein fluorescence in the milliseconds time scale by a stopped-flow apparatus. The experimental traces, obtained after rapid mixing of enzyme with increasing GSH concentrations, were well described by single exponential curves from which the apparent first order rate constants (k obs) have been calculated. The native enzyme shows k obs values (65 s−1 at 1 mm GSH and 37 °C) at least 10 times lower than those found for the Alpha, Mu, and Pi class GSTs at the same GSH concentration but at 5 °C (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar, 11Caccuri A.M. Antonini G. Board P.G. Parker M.W. Nicotra M. Lo Bello M. Federici G. Ricci G. Biochem. J. 1999; 344: 419-425Crossref PubMed Scopus (54) Google Scholar). Furthermore, unlike that observed in the more recently evolved GSTs,k obs values for hGSTT2-2 follow a linear dependence on GSH concentration within the large range of 0.12–6 mm (Fig. 3 A). These data are well described by Scheme I, which shows the binding event as a simple bimolecular interaction to give the Michaelis complexE-GSH. κonE+GSH⇔E−GSHκoff ScHEME I Linear regression analysis gives a k on = (2.7 ± 0.8) × 104m−1 s−1and a k off = 36 ± 9 s−1. Thus, a dissociation constant (K d) of about 1.3 mm for theE-GSH binary complex formation has been calculated at 37 °C (Fig. 3 A and TableI). As k on is far from the value expected for a diffusion limited process, a rapid equilibrium between at least two G-site conformations is likely. Only the less populated conformation should be competent for a proper interaction with GSH. The mutant enzyme always displaysk obs values higher (5–10-fold) than those observed for the native enzyme at the same temperature and at the same GSH concentration (Fig. 3 A). For example, at 1 mm GSH, k obs is about 500 s−1 in the R107A mutant which is 65 s−1 in the native enzyme. In addition, the dependence of the observed rate constants on GSH concentration is not linear, but now it follows a hyperbolic behavior. This kinetic trend is similar to that found for Alpha, Mu, and Pi class GSTs (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar, 11Caccuri A.M. Antonini G. Board P.G. Parker M.W. Nicotra M. Lo Bello M. Federici G. Ricci G. Biochem. J. 1999; 344: 419-425Crossref PubMed Scopus (54) Google Scholar), and it is diagnostic for a multistep binding mechanism (see Scheme II). E+GSH⇔koffkonE­GSH⇔k−2k2E*­GSH SCHEMEIIThe proposed minimal scheme describes a first fast interaction of GSH with the enzyme to give a weak pre-complex which is slowly converted into the final Michaelis complex. Only this final complex is responsible for the fluorescence perturbation at 340 nm. Nonlinear fitting of all experimental data to Scheme II gives ak on ≥ 1 × 105m−1 s−1(the angular coefficient of the tangent to the hyperbola at the lowest GSH concentrations) and a k off ≥ 140 s−1. These values are about 4-fold higher than those calculated for the native enzyme. The resulting dissociation constant for the pre-complex (k off/k on) is 1.4 mm. The microscopic rate constants for the conversion of this pre-complex into the final Michaelis complex arek 2 = 741 ± 50 s−1and k −2 = 215 ± 30 s−1. The overall dissociation constant ((k off/k on) × (k −2/k 2)) is 0.4 mm, a value lower than that observed for the native enzyme (see Table I).Table IMicroscopic rate constants for the binding of GSH to hGSTT2-2 and R107A mutantWild-typeR107Ak on(2.7 ± 0.8) × 104m−1 s−1≥1 × 105m−1 s−1k off36 ± 9 s−1≥140 s−1k off/k on1.3 mm1.4 mmk 2741 ± 50 s−1k −2215 ± 30 s−1k −2/k 20.29(k off/k on)(k −2/k 2)0.40 mmExperiments were performed at 37 °C as described under “Experimental Procedures.” Definitions of microscopic rate constants are given in Schemes I and II for the wild-type and R107A mutant, respectively. Open table in a new tab Experiments were performed at 37 °C as described under “Experimental Procedures.” Definitions of microscopic rate constants are given in Schemes I and II for the wild-type and R107A mutant, respectively. The effect of pH on the rate of GSH binding has also been analyzed. In hGSTT2-2, thek obs increases at low pH values, and at pH 5.0 (kobs = 110 s−1) it is about 4-fold higher than that observed at pH 9.0 (k obs= 29 s−1) (Fig. 3 B). This suggests that the protonation of one or more protein residues facilitates a correct interaction with the substrate. In R107A mutant, GSH binds withk obs values higher than that shown for the wild type at the same pH value but with a similar pH dependence (Fig.3 B). Thus one or more residues, but not Arg-107, are involved in the observed facilitation of GSH interaction at acidic pH values. The first observation coming from the present data is that hGSTT2-2 is able to activate GSH with an efficiency very similar to that shown by the more recently evolved GSTs. In fact, the apparent pK a for the sulfhydryl group of the bound GSH is 6.15, a value in the range of those observed for Alpha, Mu, and Pi class GSTs (pK a = 6.0–6.8). This value, obtained by a direct spectroscopic determination of the thiolate ion, is close to that calculated by kinetic experiments for rat T2-2 enzyme (13Jemth P. Mannervik B. Biochemistry. 1999; 38: 9982-9991Crossref PubMed Scopus (21) Google Scholar). Thus, these findings indicate that the substrate activation is a property acquired early by GSTs and strictly conserved during evolution. The mechanisms for activation and stabilization of the sulfhydryl atom, however, could be different among GSTs; the thiolate is hydrogen-bonded to a Tyr residue in the more recently evolved GSTs (1Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (946) Google Scholar), whereas a similar role is played by a serine residue in the insect Delta class GST previously classified as a Theta-like GST (17Caccuri A.M. Antonini G. Nicotra M. Battistoni A. Lo Bello M. Board P.G. Parker M.W. Ricci G. J. Biol. Chem. 1997; 272: 29681-29686Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Even hGSTT2-2 shows a Ser residue and not a Tyr, within hydrogen bonding of the GSH sulfhydryl group (6Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1997; 6: 309-322Abstract Full Text Full Text PDF Scopus (133) Google Scholar). The substitution of Ser-11 by Ala in the rat GSTT2-2 seems to cause an increase of the GSH pK a of 1.3 pH units (14Jemth P. Mannervik B. J. Biol. Chem. 2000; 275: 8618-8624Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). However, we noted that in the human Theta GSTT2-2, Arg-107 is in hydrogen bonding distance of the main chain carbonyl of the γ-glutamyl moiety of GSH and forms an interaction with the thiol sulfur of GSH either directly or through a water molecule (6Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1997; 6: 309-322Abstract Full Text Full Text PDF Scopus (133) Google Scholar). Subsequent mutagenesis and modeling studies suggested this residue is involved in the sulfatase reaction and in electrostatic substrate recognition (16Flanagan J.U. Rossjohn J. Parker M.W. Board P.G. Chelvanayagam G. Protein Sci. 1999; 8: 2205-2212Crossref PubMed Scopus (13) Google Scholar). The present data indicate that mutation of Arg-107 has a remarkable negative effect on the deprotonation of the substrate GSH. In R107A mutant, the apparent pK a of the bound GSH shifts from pH 6.1 to pH 7.8. Arg-107 could act both as a counterion to promote ionization of the GSH thiolate and then stabilize the thiolate by direction interaction. This is not the first case of arginine residues being implicated in GSH activation. In hGSTA1-1 (Alpha class), Arg-15 is within hydrogen bonding distance of the GSH thiol (18Sinning 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 (409) Google Scholar), and its activation/binding role was subsequently confirmed by mutagenesis (19Björnestedt R. Stenberg G. Widersten M. Board P.G. Sinning I. Jones T.A. Mannervik B. J. Mol. Biol. 1995; 247: 765-773Crossref PubMed Scopus (101) Google Scholar). In hGSTM2-2 kinetic data are consistent with Arg-107 playing a role in promoting ionization and binding of GSH (20Patskovsky Y.V. Patskovska L.N. Listowsky I. J. Biol. Chem. 2000; 275: 3296-3304Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In conclusion, on the basis of our data on the human enzyme and of those on the rat GSTT2-2 (13Jemth P. Mannervik B. Biochemistry. 1999; 38: 9982-9991Crossref PubMed Scopus (21) Google Scholar, 14Jemth P. Mannervik B. J. Biol. Chem. 2000; 275: 8618-8624Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), it is likely that GSH activation is achieved by the synergistic action of at least two residues, Arg-107 and Ser-11. As for the Alpha, Mu, and Pi GSTs, this old enzyme also extrudes quantitatively the thiol proton of GSH from the active site into the surrounding solution but only up to pH 6.5. Above this value, a protein residue acts as a base in capturing the thiol proton (see Fig. 2). The apparent pK a of this internal base is 7.3, a value that suggests the involvement of a histidine residue, possibly His-40, which is located close to the bound GSH. A nitrogen atom of the imidazole ring of His-40 is in van der Waals contact of the glycyl moiety of GSH. The capture of the thiol proton at high pH values has been also observed in the Delta GST (17Caccuri A.M. Antonini G. Nicotra M. Battistoni A. Lo Bello M. Board P.G. Parker M.W. Ricci G. J. Biol. Chem. 1997; 272: 29681-29686Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In that case His-38 and/or His-50 are probably involved. The ability to release quantitatively the thiol proton in solution at any pH value is probably an evolutionary advantage reached by the Alpha, Mu, and Pi class GSTs; during the enzymatic turnover, the proton neutralized by the protein residue in hGSTT2-2 must be released before a new productive cycle can start, and this step may limit the overall velocity. A second aspect we analyzed is the thermodynamic and kinetic efficiency of substrate binding to hGSTT2-2. This old enzyme shows a low affinity for GSH as suggested by an apparent K d of 0.8 mm, a value at least four times higher than that found in the more recently evolved GSTs. It appears that Alpha, Mu, and Pi GSTs are under an evolutionary pressure in the direction of lowerK d values. This trend is distinctive for enzymes that exhibit k cat/K m ratios far from the diffusion control limit (108–1010m−1 s−1) (21Pettersson G. Eur. J. Biochem. 1989; 184: 561-566Crossref PubMed Scopus (40) Google Scholar). Actually, all GSTs are distant from a perfectly evolved catalyst, and hGSTT2-2 shows a specificity constant for GSH of only 102m−1s−1. Moreover, a first enzyme on a metabolic pathway (like hexokinase in the glucose metabolism) often evolves toward a lower K m (or K d) value to prevent accumulation of intermediates; this could be the case of GST as the starting enzyme in the mercapturic acid formation. From a kinetic point of view, the observed rate constants for binding of GSH to the G-site (at 37 °C) are remarkably lower (about 10 times) than those observed in the Alpha, Mu, and Pi GSTs (at 5 °C) (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar, 11Caccuri A.M. Antonini G. Board P.G. Parker M.W. Nicotra M. Lo Bello M. Federici G. Ricci G. Biochem. J. 1999; 344: 419-425Crossref PubMed Scopus (54) Google Scholar). Above all, the most striking peculiarity of the Theta class enzyme is the linear dependence of k obs on GSH concentration which is consistent with a single step binding mechanism,i.e. the Michaelis complex is formed by a bimolecular encounter between GSH and enzyme. Is it possible to elucidate the evolutionary pathway GSTs have utilized to optimize their interaction with the substrate? Our data suggest a shifting from a single step binding mechanism to a multistep binding process, such as observed in Alpha, Mu, and Pi GSTs (10Caccuri A.M. Lo Bello M. Nuccetelli M. Nicotra M. Rossi P. Antonini G. Federici G. Ricci G. Biochemistry. 1998; 37: 3028-3034Crossref PubMed Scopus (58) Google Scholar, 11Caccuri A.M. Antonini G. Board P.G. Parker M.W. Nicotra M. Lo Bello M. Federici G. Ricci G. Biochem. J. 1999; 344: 419-425Crossref PubMed Scopus (54) Google Scholar). These younger GSTs are able to interact with GSH through a weak pre-complex, followed by at least one isomerization step that results in a more rapid attainment of the binding equilibrium. This strategy is reminiscent of that used by many enzymes in catalysis where a single specific reaction is normally subdivided into a number of chemical steps with lower activation energies. From a structural point of view, it is reasonable to propose that deletion of the extra C-terminal segment, which mostly obscures the G-site of the human Theta class (6Rossjohn J. McKinstry W.J. Oakley A.J. Verger D. Flanagan J. Chelvanayagam G. Tan K.-L. Board P.G. Parker M.W. Structure. 1997; 6: 309-322Abstract Full Text Full Text PDF Scopus (133) Google Scholar), should facilitate the GSH interaction. Furthermore, other important factors must be considered that refer to the dynamics of this enzyme. A plot of the crystallographic B-factors along the polypeptide chain can give an indication of the relative flexibility of a protein portion compared with other regions. As shown in Fig. 4the Alpha, Mu, and Pi GSTs display a similar and well defined flexibility pattern. Several “hot” regions with high mobility can be identified (helix 2 and its flanking regions, C-terminal segment of helix 4, loop between helices 4 and 5, and N-terminal segment of helix 5) separated by a number “cold” segments. The hGSTT2-2 enzyme shows a completely different flexibility as no distinctive hot and cold regions can be defined (the noise in T2-2 plot is due to the limited resolution of the crystal structure) (Fig. 4). It appears that GSTs have utilized flexibility in terms of an evolutionary progression. Flexibility explains some of the behavior of the R107A mutant. Arg-107 forms a salt bridge with Asp-104, and its replacement by Ala probably increases the structural flexibility of the enzyme. This is a plausible explanation for the remarkable increase of the rate of the GSH binding in the R107A mutant. In addition, replacement of Arg-107 changes the binding event from a single step to a multistep mechanism (see Fig.3 A), as observed in the more recently evolved GSTs. In other words, it appears that this specific substitution in the Theta class enzyme produces an improvement of the kinetic efficiency of GSH binding, and the mutant enzyme now resembles the behavior of the more recently evolved GSTs. We thank Prof. M. Coletta for helpful discussions.