Delineation of the structural and functional role of Arg111 in GSTU4-4 from Glycine max by chemical modification and site-directed mutagenesis

摘要: Abstract The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed site-directed mutagenesis. arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme borate buffer at pH 8.0, with pseudo-first-order saturation kinetics. rate inactivation exhibited a non-linear dependence on concentration BTD which can be described reversible binding to (KD 81.2 ± 9.2 mM) prior irreversible reaction, maximum constants 0.18 ± 0.01 min− 1. Protection afforded substrate analogues demonstrating specificity reaction. Structural analysis suggested that modified residue is Arg111, confirmed protein chemistry experiments. Site-directed mutagenesis used dissecting binding, catalytic mechanism. mutant Arg111Ala unchanged Km value for GSH but showed reduced affinity xenobiotic substrates, higher kcat specific activities towards aromatic substrates lower aliphatic substrates. biological significance dicarbonyl compounds as target engineering were discussed.