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• W2000964744 abstract "Cytosolic GSTs (glutathione S-transferases) are a major reserve of high-capacity binding proteins and exhibit ligand-binding properties for a large variety of compounds. In the present study, the binding of two non-substrate anthraquinone dyes VBAR (Vilmafix Blue A-R) and CB3GA (Cibacron Blue 3GA) to maize (Zea mays) GST I was investigated. The results showed that the enzyme was specifically and irreversible inactivated by VBAR with a Kd of 35.5±2.2 μM and a k3 of 0.47 min−1. Proteolytic cleavage of the VBAR-modified enzyme and subsequent separation of peptides gave only one modified peptide. Sequencing of the modified peptide revealed the target site of VBAR reaction to be Lys41. CB3GA binds reversibly to GST I and behaves as a competitive inhibitor towards CDNB (1-chloro-2,4-dinitrobenzene) and glutathione. CB3GA binding to GST I is accompanied by a characteristic spectral change in the absorption at positive maximum (670 nm) which exhibited a hyperbolic dependence on dye concentration with a Kd of 12.1±0.5 μM. Site-directed mutagenesis of selected residues (Trp12, Phe35, Lys41, Asn49, Gln53, Ser67 and Ile118) was employed, and the mutated enzymes were assessed for CB3GA binding. These results, together with molecular-modelling studies, established that the ligandin-binding site of GST I is located mainly in the hydrophobic binding site. The ability of VBAR to specifically inactivate GST I was exploited further to demonstrate the specific binding of several plant hormones and flavonoids to GST I. The inactivation of other GST isoenzymes by VBAR was also investigated, and it was concluded that VBAR may have wide applicability as an affinity label for probing structure–function relationships of GST isoenzymes." @default.
• W2000964744 created "2016-06-24" @default.
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• W2000964744 date "2004-09-07" @default.
• W2000964744 modified "2023-10-16" @default.
• W2000964744 title "Characterization of the ligandin site of maize glutathione S-transferase I" @default.
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• W2000964744 doi "https://doi.org/10.1042/bj20040298" @default.
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