Glutathione Transferase: New Model for Glutathione Activation

Abstract

Glutathione transferases are enzymes of the cellular detoxification system that metabolize a vast spectrum of xenobiotic and endobiotic toxic compounds. They are homodimers or heterodimers and each monomer has an active center composed of a G-site in which glutathione (GSH) binds and an H-site for the electrophilic substrate. When GSH binds to the G-site, the pKa value of its thiol group drops by 2.5 units; this promotes its deprotonation and, therefore, produces a strong nucleophilic thiolate that is able to react with the electrophilic substrate. The mechanism behind the deprotonation of the thiol group is still unknown. Some studies point to the fact that the GSH glutamyl alpha-carboxylate group is essential for GSH activation, whereas others indicate the importance of the active-center water molecules. On the basis of QM/MM calculations, we propose a mechanism of GSH activation in which a water molecule, acting as a bridge, is able to assist in the transfer of the proton from the GSH thiol group to the GSH glutamyl alpha-carboxylate group, after an initial GSH conformational rearrangement. We calculated the potential of mean force of this GSH structural rearrangement that would be necessary for the approach of both groups and we then performed a QM/MM ONIOM scan of water-assisted proton transfer. The overall free-energy barrier for the process is consistent with experimental studies of the enzyme kinetics.

A Glutathione Transferase from Agrobacterium tumefaciens Reveals a Novel Class of Bacterial GST Superfamily

“…In the Atu GSTH1-1/Nb-GSH complex, the conserved residues Arg34, Glu85, Ser86, Gln68 and Asn120' appear to form the proposed electron-sharing network. Based on Quantum mechanics/Molecular mechanics (QM/MM) calculations it was recently proposed [37] that the GSH activation by GSTs is accomplished by a water-assisted proton-transfer mechanism that takes into account the suggested roles of the GSH γ-glutamyl carboxylate group and the active-site water molecules. According to this mechanism, a water molecule acting as a bridge is able to transfer the proton from the GSH thiol group to the GSH γ-glutamyl carboxylate group.…”

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

“…The pK a of the α-carboxylate, in turn, is undoubtedly lowered by the side-on, out-of-plane interaction with the guanidinium of Arg-38 (torsion angle 45.9°), which is itself engaged in solvent-mediated interactions with the main-chain carboxyl groups of Ala-43 and Arg-60. This architecture is highly reminiscent of the "electron-sharing network" that is functionally conserved in all classes of soluble GSTs for the same purpose (32), and the use of a bridging water molecule to transfer the thiol proton to the α-carboxylate of GSH has been shown to be energetically favorable within an alpha class soluble GST (33). Crucially, density for this water is absent for the Phenix (34) refined bis-phenyl GSH complex (PDB ID code 4AL1), in which the relative occupancies to GSH were refined as 0.87:0.13, respectively.…”

The fungal glutathione S-transferase system. Evidence of new classes in the wood-degrading basidiomycete Phanerochaete chrysosporium

“…Furthermore, in the URE2p class, an Asn seems to be essential for the activation of the thiolate of GSH [19]. In addition, a model for GSH activation which involves a water molecule and the GSH glutamyl a-carboxylate group has been proposed [20].…”