On the reaction mechanism of class Pi glutathione S-transferase

Abstract

Theoretical calculations were performed to examine the ionization of the phenolic group of Tyr7 and the thiol group of glutathione in aqueous solution and in the protein class-pi glutathione S-transferase (GST-Pi). Three model systems were considered for simulations in the protein environments the free enzyme, the complex between glutathione and the enzyme, and the complex between 1-chloro-2.4-dinitrobenzene, glutathione, and the enzyme. The structures derived from Molecular Dynamics simulations were compared with the crystallographic data available for the complex between the inhibitor S-(p-nitrobenzyl)glutathione and GST-Pi, the glutathione-bound form of GST-Pi, and the free enzyme carboxymethylated in Cys47. Free-energy perturbation techniques were used to determine the thermodynamics quantities for ionization of the phenol and thiol groups. The functional implications of Tyr7 in the activation of the glutathione thiol group are discussed in the light of present results, which in agreement with previous studies suggest that Tyr7 in un-ionized form contributes to the catalytic process of glutathione S-transferase, the thiolate anion being stabilized by hydrogen bond with Tyr7 and by interactions with hydrating water molecules.

Site‐directed mutagenesis of mouse glutathione transferase P1‐1 unlocks masked cooperativity, introduces a novel mechanism for ‘ping pong’ kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione

“…This gives an indication of the nature of the active site at various points during the reaction. Molecular dynamic studies have suggested that GS − is stabilized by the OH group of Y7 (tyrosine) and the water molecules W1, W2 and W4, and is consistent with a model whereby the proton from glutathione is initially displaced onto water molecule W0 [38,39]. The structure of the C47A mutant of mGSTP1‐1 complexed with S‐ ( p ‐nitrobenzyl)glutathione presented here reveals an intriguing difference in alignment of the inhibitor at the two sites.…”

“…In addition, the water channel identified in human GSTP1 [41] and rat, rattus norvegicus GSTA1 [42] as connecting the two active sites of the dimer may also play a functional role in the observed cooperativity. Finally, there have been a number of suggestions as to the ultimate proton sink, including a channel filled by four water molecules, which passes between helices αA and αF and reaches the solvent at R11 [32], the α‐carboxylate of the γ‐glutamyl residue of GSH [43], or simply extrusion of W0 [38,39].…”