Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues

Abstract

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247-250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with l-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of l-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.

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

“…CDNB as Substrate-Despite previous reports (13) in our experimental conditions, we found that hGSTT2-2 has a small but detectable activity with CDNB, one of the best co-substrates for Alpha, Pi, and Mu GSTs. The presence of phosphate seems to be crucial for this activity, and at pH 6.5 and 37°C, hGSTT2-2 displays a k cat value of 0.07 s Ϫ1 and a K m(GSH) of about 0.6 mM.…”

“…GSH, S-hexylglutathione, CDNB, and 1-fluoro-2,4-dinitrobenzene (FDNB) were Sigma products. His-tagged recombinant hGSTT2-2 and R107A mutant were expressed in Escherichia coli and purified using immobilized metal ion chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen) as described previously (13,17).…”

“…Most of the kinetic studies have been carried out using 1-menaphthyl sulfate (Msu) as co-substrate. A few experiments utilized 1-chloro-2,4-dinitrobenzene (CDNB), one of the best co-substrates for the Alpha, Pi, and Mu GSTs but which was previously considered not to be a substrate for the Theta enzyme (13). The crystallographic studies demonstrated that the crystals were catalytically active in that they could convert the sulfatase substrate, Msu, into the corresponding GSH conjugate with cleavage of the sulfate group.…”

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of Substrate Binding to the Active Site of Glutathione Transferases

“…His-tagged recombinant GST2-2 and R107A mutants were expressed in Escherichia coli and purified using immobilized metal ion chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen) as described previously (12,16).…”

“…Does such a mechanism hold for the primitive GSTT2-2? Furthermore, all GSTs activate the substrate by lowering the pK a value of GSH at the active site, but the peculiar sulfatase reaction catalyzed by hGSTT2-2 could not need the thiolate form of GSH (12). Interestingly, in this old enzyme, Ser-11 replaces the Tyr residue found in Alpha, Pi, and Mu class GSTs.…”