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

Tài liệu tham khảo về hóa-sinh bằng tiếng anh | ỊFEBS Journal Different roles of functional residues in the hydrophobic binding site of two sweet orange tau glutathione S-transferases Angela R. Lo Piero Valeria Mercurio Ivana Puglisi and Goffredo Petrone Dipartimento di Scienze Agronomiche Agrochimiche e delle Produzioni Animali DACPA University di Catania Italy Keywords glutathione S-transferase H-site site-directed mutagenesis sweet orange tau class glutathione S-transferase Correspondence A. R. Lo Piero Dipartimento di Scienze Agronomiche Agrochimiche e delle Produzioni Animali DACPA Universita di Catania Via S. Sofia 98 95123 Catania Italy Fax 39 95 7141581 Tel 39 95 7580238 E-mail rlopiero@ Received 27 July 2009 revised 7 October 2009 accepted 5 November 2009 doi Glutathione S-transferases GSTs catalyze the conjugation of glutathione to hydrophobic compounds contributing to the metabolism of toxic chemicals. In this study we show that two naturally occurring tau GSTs GSTUs exhibit distinctive kinetic parameters towards 1-chloro-2 4-dini-trobenzene CDNB although they differ only in three amino acids Arg89 Glu117 and Ile172 in GSTU1 are replaced by Pro89 Lys117 and Val172 in GSTU2 . In order to understand the effects of the single mismatched residues several mutant GSTs were generated through site-directed mutagenesis. The analysis of the kinetic parameters of the mutants led to the conclusion that Glu117 provides a critical contribution to the maintenance of a high-affinity CDNB-binding site. However the substitution E117K gives rise to mutants showing increased kcat values for CDNB suggesting that Lys117 might positively influence the formation of the transition state during catalysis. No changes in the Km values towards glutathione were found between the naturally occurring GSTs and mutants except for the mutant caused by the substitution R89P in GSTU1 which showed a sharp increase in Km. Moreover the analysis of enzyme reactivation after denaturation showed that .