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.