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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 34, Number 9
BKCSDE 34(9)
September 20, 2013 

 
Title
Site-directed Mutagenesis of the Evolutionarily Conserved Tyr8 Residue in Rice Phi-class Glutathione S-transferase F3
Author
Hyun-Joo Jo, Mi-Jin Pack, Jin-Young Seo, Jin-Kyung Lim, Kwang-Hoon Kong*
Keywords
Tyrosine 8 residue, Rice, Glutathione S-transferase, Site-directed mutagenesis, Kinetic parameters
Abstract
To elucidate the role of the evolutionarily conserved Tyr8 residue in rice Phi-class GSTF3, this amino acid was replaced with alanine and phenylalanine by site-directed mutagenesis, respectively. The replacement of Tyr8 with Ala significantly affected the catalytic activity and the kinetic parameters, whereas the substitutions of Tyr8 with Phe had almost no effect. The Y8A mutant resulted in approximately 90-100% decrease of the specific activity. Moreover, the Y8A mutant resulted approximately in 2-fold increase of Km, approximately 60-80% decrease of kcat, and approximately 6.5-fold decrease in kcat/Km. From the pH/log kcat/Km plot, pKa values of the GSH in the wild-type enzyme-GSH complex, Y8A-GSH complex and Y8F-GSH complex were estimated to be approximately 6.8, 8.5 and 6.9, respectively. From these results, we suggest that the evolutionarily conserved Tyr8 residue in OsGSTF3 seems to influence the structural stability of the active site of OsGSTF3 rather than directly its catalytic activity.
Page
2671 - 2674
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