Abstract
In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-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 alpha-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 pKa 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.
Highlights
Glutathione transferases (GSTs,1 EC 2.5.1.18) are a superfamily of multifunctional enzymes involved in the cellular detoxification of various physiological and xenobiotic substances [1,2,3,4]
Vided into steps involving binding of substrates to the enzyme active site, activation of GSH by thiol deprotonation, nucleophilic attack by the thiolate at the electrophilic center, product formation, and product release (8 –11)
A step of the activation of GSH by deprotonation of the thiol group to enhance the nucleophilicity for reaction with diverse types of electrophilic centers is crucial for the enzymatic catalysis
Summary
Glutathione transferases (GSTs, EC 2.5.1.18) are a superfamily of multifunctional enzymes involved in the cellular detoxification of various physiological and xenobiotic substances [1,2,3,4]. Vided into steps involving binding of substrates to the enzyme active site, activation of GSH by thiol deprotonation, nucleophilic attack by the thiolate at the electrophilic center, product formation, and product release (8 –11). A step of the activation of GSH by deprotonation of the thiol group to enhance the nucleophilicity for reaction with diverse types of electrophilic centers is crucial for the enzymatic catalysis. Apart from the foregoing, several experimental observations have implicated an alternative mechanism named “base-assisted deprotonation” [25, 27] This model has been proposed on the basis of the function of the glutamyl ␣-carboxylate of glutathione as a catalytic base that accepts the thiol proton from the thiol group. Introduction of a carboxylate in the glutathione binding site of the enzyme in a location generally occupied by the glutamyl ␣-carboxylate partially res-
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