Non-active site residues Cys69 and Asp150 affected the enzymatic properties of glutathione S-transferase AdGSTD3-3
| Autor: | Jeerang Wongtrakul, Albert J. Ketterman, Rungrutai Udomsinprasert |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Stereochemistry Amino Acid Motifs Biochemistry Catalysis Substrate Specificity Protein structure Anopheles Enzyme Stability Animals Cysteine Enzyme Inhibitors Binding site Site-directed mutagenesis Structural motif Molecular Biology Glutathione Transferase chemistry.chemical_classification Aspartic Acid Binding Sites biology Chemistry Mutagenesis Active site Recombinant Proteins Enzyme assay Protein Structure Tertiary Isoenzymes Kinetics Enzyme Amino Acid Substitution Insect Science Mutagenesis Site-Directed biology.protein Dimerization |
| Zdroj: | Insect Biochemistry and Molecular Biology. 33:971-979 |
| ISSN: | 0965-1748 |
| DOI: | 10.1016/s0965-1748(03)00103-6 |
| Popis: | To elucidate how non-active site residues support the catalytic function, five selected residues of AdGSTD3-3 isoenzyme were changed to AdGSTD1-1 residues by means of site-directed mutagenesis. Analysis of the kinetic parameters indicated that Cys69Gln and Asp150Ser showed marked differences in Vmax and Km compared with the wild type enzyme. Both residues were characterized further by replacement with several amino acids. Both the Cys69 and Asp150 mutants showed differences with several GST substrates and inhibitors including affecting the interactions with pyrethroid insecticides. Cys69 and Asp150 mutants possessed a decreased half-life relative to the wild type enzyme. The Asp150 mutation appears to affect neighboring residues that support two important structural motifs, the N-capping box and the hydrophobic staple motif. The Cys69 mutants appeared to have subtle conformational changes near the active site residues resulting in different conformations and also directly affecting the active site region. The results show the importance of the cumulative effects of residues remote from the active site and demonstrate that minute changes in tertiary structure play a role in modulating enzyme activity. |
| Databáze: | OpenAIRE |
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