Characterization of the ligandin site of maize glutathione S-transferase I.
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 K(d) of 35.5+/-2.2 microM and a k(3) 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 Lys(41). 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 K(d) of 12.1+/-0.5 microM. Site-directed mutagenesis of selected residues (Trp(12), Phe(35), Lys(41), Asn(49), Gln(53), Ser(67) and Ile(118)) 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.
Axarli
Irine A
IA
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855-Athens, Greece.
Rigden
Daniel J
DJ
Labrou
Nikolaos E
NE
D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D005419:Flavonoids / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010937:Plant Growth Regulators / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D014227:Triazines / Q000378:metabolism / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology*
D000880:Anthraquinones; D005419:Flavonoids; D007527:Isoenzymes; D010937:Plant Growth Regulators; D014227:Triazines; C091762:Vilmafix Blue A-R; C012842:Cibacron Blue F 3GA; D005982:Glutathione Transferase