title: The catalytic Tyr-9 of glutathione S-transferase A1-1 controls the dynamics of the C terminus.
abstract: The glutathione S-transferase enzymes (GSTs) have a tyrosine or serine residue at their active site that hydrogen bonds to and stabilizes the thiolate anion of glutathione, GS(-). The importance of this hydrogen bond is obvious, in light of the enhanced nucleophilicity of GS(-) versus the protonated thiol. Several A-class GSTs contain a C-terminal segment that undergoes a ligand-dependent local folding reaction. Here, we demonstrate the effects of the Y9F substitution on binding affinity for glutathione conjugates and on rates of the order-disorder transition of the C terminus in rat GST A1-1. The equilibrium binding affinity of the glutathione conjugate, GS-NBD (NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole), was decreased from 4.09 microm to 0.641 microm upon substitution of Tyr-9 with Phe. This result was supported by isothermal titration calorimetry, with K(d) values of 1.51 microm and 0.391 microm for wild type and Y9F, respectively. The increase in binding affinity for the mutant is associated with dramatic decreases in rates for the C-terminal order-disorder transition, based on a stopped-flow kinetic analysis. The same effects were observed, qualitatively, for a second GSH conjugate, GS-ethacrynic acid. Apparently, the phenolic hydroxyl group of Tyr-9 is critical for orchestrating C-terminal dynamics and efficient product release, in addition to its role in lowering the pK(a) of GSH.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Nieslanik
authors.forename: W M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D020134:Catalytic Domain; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010649:Phenylalanine; D011487:Protein Conformation; D051381:Rats; D012694:Serine; D013816:Thermodynamics; D014443:Tyrosine
chemical_list: D007527:Isoenzymes; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D005982:Glutathione Transferase; D009327:4-Chloro-7-nitrobenzofurazan; D005978:Glutathione