International Journal of Biological Macromolecules 32 (2003) 67–75 Role of mutation Y6F on the binding properties of Schistosoma japonicum glutathione S-transferase Zeyad Yassin, Emilia Ortiz-Salmerón, M. José Clemente-Jiménez, Carmen Barón, Luis Garc´ ıa-Fuentes ∗ Dpto. de Qu´ ımica F´ ısica, Bioqu´ ımica y Q. Inorgánica, Facultad de Ciencias Experimentales, Universidad de Almer´ ıa, La Cañada de San Urbano, 04120 Almer´ ıa, Spain Received 17 September 2002; received in revised form 7 January 2003; accepted 3 March 2003 Abstract The role of the hydroxyl group of tyrosine 6 in the binding of Schistosoma japonicum glutathione S-transferase has been investigated by isothermal titration calorimetry (ITC). A site-specific replacement of this residue with phenylalanine produces the Y6F mutant, which shows negative cooperativity for the binding of reduced glutathione (GSH). Calorimetric measurements indicated that the binding of GSH to Y6F dimer is enthalpically driven over the temperature range investigated. A concomitant net uptake of protons upon binding of GSH to Y6F mutant was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The entropy change is favorable at temperatures below 26 ◦ C for the first site, being entropically favorable at all temperatures studied for the second site. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative C ◦ p1 =-3.45 ± 0.62 kJ K -1 mol -1 for the first site, whereas a small C ◦ p2 =-0.33 ± 0.05 kJ K -1 mol -1 for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate. © 2003 Elsevier B.V. All rights reserved. Keywords: Glutathione S-transferase; Schistosoma japonicum; Glutathione; Unfolding; Fluorescence; Binding; Microcalorimetry; S-Methylglutathione 1. Introduction The glutathione S-transferases (GSTs) are a family of dimeric detoxication enzymes, which catalyze the conjuga- tion of GSH to a variety of endogenous and exogenous elec- trophiles. The mammalian cytosolic GSTs are separated into seven gene classes based on crystal structure and substrate specificity: alpha (), pi (), mu (), theta (), kappa (), sigma (), and zeta () [1–5]. X-ray crystallographic and site-directed mutagenesis studies illustrate that each GST contains a conserved tyrosine or serine residue which hy- drogen bonds to, and effectively deprotonates, GSH to the nucleophilic thiolate (GS) [6–9]. Because the thiolate anion is a more reactive nucleophile than the protonated thiol, the Abbreviations: GSH, reduced glutathione; Y6F, glutathione S-transferase Y6F mutant from Schistosoma japonicum; DTT, dithio- threitol; CDNB, 1-chloro-2,4-dinitrobenzene; ITC, isothermal titra- tion calorimetry; Mops, 3-(N-morpholino) propanesulfonic acid; Aces, 2-[(2-amino-2-oxoethyl)-amino]ethanesulfonic acid ∗ Corresponding author. Tel.: +34-950-015618; fax: +34-950-015008. E-mail address: lgarcia@ual.es (L. Garc´ ıa-Fuentes). catalytic advantage is obvious [10]. Crystal structures indi- cate that each GST has the same basic protein fold, which consists of two domains; one domain provides the binding site for GSH (G-site), and the other contributes to the binding of the hydrophobic ligand (H-site) [11]. Although the G-site is highly homologous in all of GSTs, there is a great deal of variability in the H-site, which confers the differing sub- strate selectivity and catalytic properties for each gene class. Site-directed mutagenesis studies have revealed the im- portance of specific residues in the glutathione binding site. Manoharan et al. [12] showed that the substitution of binding site residues Arg 13, Gln 62, and Asp 96 in  GST resulted in 20–50-fold decreases of both the catalysis and glutathione binding efficiencies in comparison to the wild-type. However, the substitution of Tyr 7 by phenylala- nine still resulted in 27% of the wild-type capacity to bind glutathione, whilst the enzymatic catalysis was reduced to less than 1%. This substantial reduction in the catalytic ac- tivity due to the substitution of tyrosine was also obtained for  GST [7]. Furthermore, there are different views on how the thiol anion of glutathione is created to promote high 0141-8130/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0141-8130(03)00039-4