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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 33, Number 12
BKCSDE 33(12)
December 20, 2012

Title	Site-directed Mutagenesis of Cysteine Residues in Phi-class Glutathione S-transferase F3 from Oryza sativa
Author	Hyun-Joo Jo, Ju-Won Lee, Jin-Seok Noh, Kwang-Hoon Kong*
Keywords	Cysteine residues, Rice, Glutathione S-transferase, Substrate specificity, Kinetic parameters
Abstract	To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the Km CDNB value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site’s conformation than in the substrate binding directly.
Page	4169 - 4172
Full Text	PDF

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