Involvement of the Carboxyl Groups of Glutathione in the Catalytic Mechanism of Human Glutathione Transferase A1-1

Abstract

The present study proposes the participation of both carboxylate groups of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional studies in combination with structural data provide evidence for the alpha-carboxylate of the Glu residue of glutathione acting as a proton acceptor in the catalytic mechanism. The Glu carboxylate is hydrogen-bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu alpha-carboxylate of glutathione is bound in a similar manner to the active sites of mammalian glutathione transferases of classes Alpha, Mu, and Pi, for which three-dimensional structures are known. Mutation of the hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of the Glu residue of glutathione (Thr68->Val) caused a shift of the pH dependence of the enzyme-catalyzed reaction, suggesting that the acidic limb of the pH-activity profile reflects the ionization of the carboxylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two Arg side chains in GST A1-1. One of these residues (Arg45) may influence an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix as a lid over the active site. Removal of the Gly residue from the glutathione molecule caused a 13-fold increase in the KM value for the electrophilic substrate. Thus, the Gly carboxylate of glutathione, by way of influencing the topology of the active site, contributes to the binding of the second substrate of the enzyme. Consequently, the glutathione molecule has several functions in the glutathione transferase catalyzed reactions, not only as a substrate providing the thiol group for different types of chemical reactions but also as a substrate contributing a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second substrate to the enzyme.

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

“…As a whole, several aspects seem to characterize the hGSTT2-2 steady state kinetics when compared with other human GSTs as follows: (a) very low (at least 100-fold lower) k cat and k cat /K m(GSH) values; (b) the apparent negative cooperativity, also observed in the Alpha and Mu GSTs, but in those enzymes it is probably the consequence of a steady state random kinetic mechanism (24); (c) k cat value is pH independent, whereas in human Alpha, Mu, and Pi GSTs (with CDNB as co-substrate) it parallels the deprotonation of the bound GSH (11,25,26).…”

The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

“…o-CF 3 CDNB is a CDNB analogue that is monitored the same way as CDNB but which gives improved saturation kinetics (35). Similarly, inhibition of the isomerization reaction by GSHex was measured in a series of reactions where AD was varied from 20 to 300 M in 42 measurements at 5 mM of GSH and different concentrations of GSHex (0, 2, 4, and 8 M).…”

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

“…[64][65][66] Hence, an increase in mutation rates favoring a given enzymatic activity will result in increased conformational heterogeneity, allowing the same enzyme to accommodate different substrates. 67,68 We have explored the conformational dynamics of three AgGSTE variants (AgGSTE2, AgGSTE2-I114T/F120L and AgGSTE5) in an attempt to identify the molecular basis of their distinct DDTase activity. The present simulations show that in presence of GSH, the three variants undergo a sequence of conformational rearrangements involving helices H4, H5 and H9 which lead either to the occlusion of the G-site by helix H2, or the opening of the G-site due to the conformational rearrangement of the C-terminal region into an antiparallel β-sheet with the C-terminus of helix H4.…”

“…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. 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).…”

“…In the base-assisted deprotonation model, the glutamyl α-carboxylate of GSH acts as the catalytic base deprotonating the thiol group. 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.…”