title: Delineation of the structural and functional role of Arg111 in GSTU4-4 from Glycine max by chemical modification and site-directed mutagenesis.
abstract: The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme in borate buffer at pH8.0, with pseudo-first-order saturation kinetics. The rate of inactivation exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of reagent to the enzyme (KD 81.2±9.2mM) prior to the irreversible reaction, with maximum rate constants of 0.18±0.01min(-1). Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in substrate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged Km value for GSH but showed reduced affinity for the xenobiotic substrates, higher kcat and specific activities towards aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xenobiotic substrate specificity were discussed.
authors.affiliation: Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece. Electronic address: lambrou@aua.gr.
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942 Alkharj, KSA, Saudi Arabia; Department of Microbiology, College of Science, Al-Azhar University, Nasr City, 11884 Cairo, Egypt.
authors.forename: Magdy Mohamed
authors.initials: MM
authors.lastname: Muharram
authors.affiliation: Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942 Alkharj, KSA, Saudi Arabia; Department of Pharmacognosy, College of Pharmacy, Alexandria University, Alexandria 21215, Egypt.
authors.forename: Maged Saad
authors.initials: MS
authors.lastname: Abdelkader
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D005978:Glutathione / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011487:Protein Conformation; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism
chemical_list: D015262:Xenobiotics; D001120:Arginine; D005982:Glutathione Transferase; D005978:Glutathione; D005998:Glycine
keywords: Chemical modification; Glutathione transferase; Molecular modelling; Site-directed mutagenesis