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STRUCTURAL AND CATALYTIC ROLE OF TWO CONSERVED TYROSINES IN D ELTA‐CLASS GLUTATHIONE S ‐TRANSFERASE FROM L ocusta migratoria
Author(s) -
Zhang Xueyao,
Li Tao,
Zhang Jianqin,
Li Daqi,
Guo Yaping,
Qin Guohua,
Zhu Kun Yan,
Ma Enbo,
Zhang Jianzhen
Publication year - 2012
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.21025
Subject(s) - tyrosine , active site , enzyme kinetics , biochemistry , stereochemistry , binding site , thermostability , mutant , biology , enzyme , substrate (aquarium) , transferase , chemistry , ecology , gene
GlutathioneS ‐transferases ( GST s) are an important family of detoxifying enzymes and play a key role in pesticide resistance in the insect. Tyrosine is essential for its detoxification function. In the present study, two conserved tyrosine residues are located at positions 108 and 116 inH ‐site ofL m GSTD 1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants , Y 108A,Y 108E,Y 116A, andY 116E, were generated . It was found that the four mutants affected the specific activity ofL m GSTD 1 in various degrees, depending on the types of substrate and reaction mechanism. Steady‐state kinetics assay revealed thatY 108E andY 116E had a significant influence onGSH ‐binding ability, which indicates the two tyrosine residues ofH ‐site contribute to topology rearrangement ofG ‐site. BothY 116A andY 116E exhibited lowerCDNB ‐binding affinity, suggesting thatY 116 takes part in hydrophobic substrate binding. The thermostability assay, intrinsic, and 8‐anilino‐1‐naphthalenesulfonic acid ( ANS ) florescence results showed that the two tyrosine residues were involved in regulation of active‐site conformation. Finally, homology modeling provided evidence that the two tyrosines inH ‐site participate in hydrophobic substrate binding. Furthermore,Y 108 is closer to theSatom ofS ‐hexylglutathione. In conclusion, the two tyrosines inL m GSTD 1 are important residues in both the catalytic process and protein stability .