Site-directed Mutagenesis of the Evolutionarily Conserved Tyr8 Residue in Rice Phi-class Glutathione S-transferase F3

Bulletin of the Korean chemical society

Hyun-Joo Jo¹  Kwang-Hoon Kong¹  Jin-Young Seo¹  Mi-Jin Pack¹  Jin-Kyung Lim¹ 

关键词: Tyrosine 8 residue;    Rice;    Glutathione S-transferase;    Site-directed mutagenesis;    Kinetic parameters;   

DOI  :  

学科分类：化学（综合）

来源: Korean Chemical Society

【 摘 要 】

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.

【 授权许可】

Unknown