Different roles of functional residues in the hydrophobic binding site of two sweet orange tau

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Different roles of functional residues in the hydrophobic binding site of two sweet orange tauDifferent roles of functional residues in the hydrophobicbinding site of two sweet orange tau glutathioneS-transferasesAngela R. Lo Piero, Valeria Mercurio, Ivana Puglisi and Goffredo Petrone `Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali (DACPA), Universita di Catania, Italy Glutathione S-transferases (GSTs) catalyze the conjugation of glutathioneKeywordsglutathione S-transferase; H-site; to hydrophobic compounds, contributing to the metabolism of toxicsite-directed mutagenesis; sweet orange; chemicals. In this study, we show that two naturally occurring tau GSTstau class glutathione S-transferase (GSTUs) exhibit distinctive kinetic parameters towards 1-chloro-2,4-dini- trobenzene (CDNB), although they differ only in three amino acidsCorrespondence (Arg89, Glu117 and Ile172 in GSTU1 are replaced by Pro89, Lys117 andA. R. Lo Piero, Dipartimento di Scienze Val172 in GSTU2). In order to understand the effects of the single mis-Agronomiche, Agrochimiche e delle ` matched residues, several mutant GSTs were generated through site-direc-Produzioni Animali (DACPA), Universita diCatania, Via S. Sofia 98, 95123, Catania, ted mutagenesis. The analysis of the kinetic parameters of the mutantsItaly led to the conclusion that Glu117 provides a critical contribution to theFax: +39 95 7141581 maintenance of a high-affinity CDNB-binding site. However, the substitu-Tel: +39 95 7580238 tion E117K gives rise to mutants showing increased kcat values forE-mail: rlopiero@unict.it CDNB, suggesting that Lys117 might positively influence the formation of the transition state during catalysis. No changes in the Km values(Received 27 July 2009, revised 7 October towards glutathione were found between the naturally occurring GSTs2009, accepted 5 November 2009) and mutants, except for the mutant caused by the substitution R89P indoi:10.1111/j.1742-4658.2009.07481.x GSTU1, which showed a sharp increase in Km. Moreover, the analysis of enzyme reactivation after denaturation showed that this R89P substitution leads to a two-fold enhancement of the refolded enzyme yield, suggesting that the insertion of proline might induce critical structural modifications. In contrast, the substitution P89R in GSTU2 does not modify the reacti- vation yield and does not impair the affinity of the mutant for glutathi- one, suggesting that all three residues investigated in this work are fundamental in the creation of enzymes characterized by unique biochem- ical properties.IntroductionThe glutathione S-transferases (GSTs; EC 2.5.1.18) are origin [1–4]. GSTs are widely distributed in naturemembers of a multifunctional superfamily of enzymes from humans to bacteria [5–7]. In plants, the GSHcatalyzing the conjugation of glutathione (GSH) to the addition reaction is coupled to the vacuolar compart-electrophilic groups of hydrophobic and usually mentation of the GSH conjugates because of the lackcytotoxic molecules of either endogenous or exogenous of an effective excretion pathway, which is active inAbbreviations4-NPB, 4-nitrophenethyl bromide; CDNB, 1-chloro-2,4-dinitrobenzene; ECA, ethacrynic acid; GmGSTU-4-4, Glycine max tau glutathioneS-transferase-4-4; GSH, glutathione; G-site, glutathione-binding site; GST, glutathione S-transferase; GSTU, tau glutathione S-transferase;H-site, hydrophobic binding site; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. 255FEBS Journal 277 (2010) 255–262 ª 2009 The Authors Journal compilation ª 2009 FEBSFunctional role of the GST H-site residues A. R. Lo Piero et al.animals [8,9]. GSTs, in addition to transfer of GSH to negatively and positively charged amino acids stabi-toxic compounds, act as GSH-dependent peroxidase, lized by an array of hydrogen bonds, and appears toisomerase and oxidoreductases, playing pivotal roles in be a functionally conserved motif in all GST classesplant cell protection, as reviewed by Moons [10]. Plant [27]. In the Glycine max GSTU-4-4 (GmGSTU4-4)–GSTs are abundantly expressed, and show major tran- GSH complex, the strictly conserved residues Arg18,scriptional regulation. It has been reported that GST Glu66, Ser67 and Asp103 appear to fo ...