An Electron-sharing Network Involved in the Catalytic Mechanism Is Functionally Conserved in Different Glutathione Transferase Classes

Abstract

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl ␣-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl ␣-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pK a of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.

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 Role of the Conformational Dynamics of Glutathione S-Transferase Epsilon Class on Insecticide Resistance inAnopheles gambiae

“…69 In the X-ray structure of AgGSTE2, the thiol group of GSH is within hydrogen-bond distance of the hydroxyl group of Ser12, which is thought to be required for the correct orientation and stabilization of the deprotonated thiolate anion in the active site (Figure 1). 19,65,66 Early in the simulations of AgGSTE2-I114T/F120L and AgGSTE5, the thiol group moves away from Ser12 and approaches the carboxylate group of Glu116 (Figure 8). This conformational change is more noticeable in the subunit where the S2-H2 loop and H4 helix shut the G-site as opposed to the subunit where the G-site remains more exposed to the solvent (Figure 7).…”

“…65, 68 A variation of this model has been proposed for the catalytic mechanism of Delta class GSTs. 18,19 In the model known as base-assisted deprotonation the glutamyl α-carboxylate of GSH acts as the catalytic base deprotonating the thiol group. 65, 68 On the basis of the structural similarity and evolutionary proximity between the Delta and Epsilon classes, the proposed mechanism is also plausible for AgGSTE2 and AgGSTE5.…”

“…19,[64][65][66][67] The catalytic activity of GSTs relies on the enzyme ability to activate GSH by lowering the pKa of its thiol group from 9.0 to values between 6.0 and 6.9. 65 It enhances the rate of the nucleophilic attack of GSH towards electrophilic co-substrates in 200 to 300-fold.…”

“…14,17 Previous structural and biochemical characterization of the Delta class GSTD4 from A. gambiae has underlined the importance of several residues for catalysis and substrate specificity of the enzyme through structural rearrangements. 18,19 These conformational changes have been postulated to alter the flexibility of the protein, and thus modulate its catalytic aptness. 1 The enzyme AgGSTE2 is thought to be the most important GST conferring DDT resistance in A. gambiae.…”