Abstract
Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.