title: Structural and catalytic role of two conserved tyrosines in Delta-class glutathione S-transferase from Locusta migratoria.
abstract: Glutathione S-transferases (GSTs) 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 in H-site of LmGSTD1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants, Y108A, Y108E, Y116A, and Y116E, were generated. It was found that the four mutants affected the specific activity of LmGSTD1 in various degrees, depending on the types of substrate and reaction mechanism. Steady-state kinetics assay revealed that Y108E and Y116E had a significant influence on GSH-binding ability, which indicates the two tyrosine residues of H-site contribute to topology rearrangement of G-site. Both Y116A and Y116E exhibited lower CDNB-binding affinity, suggesting that Y116 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 in H-site participate in hydrophobic substrate binding. Furthermore, Y108 is closer to the S atom of S-hexylglutathione. In conclusion, the two tyrosines in LmGSTD1 are important residues in both the catalytic process and protein stability.
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, China.
authors.forename: Xueyao
authors.initials: X
authors.lastname: Zhang
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Jianqin
authors.initials: J
authors.lastname: Zhang
authors.forename: Daqi
authors.initials: D
authors.lastname: Li
authors.forename: Yaping
authors.initials: Y
authors.lastname: Guo
authors.forename: Guohua
authors.initials: G
authors.lastname: Qin
authors.forename: Kun Yan
authors.initials: KY
authors.lastname: Zhu
authors.forename: Enbo
authors.initials: E
authors.lastname: Ma
authors.forename: Jianzhen
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D048188:Locusta migratoria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D040681:Structural Homology, Protein
chemical_list: D019476:Insect Proteins; D005982:Glutathione Transferase