Catalytically active monomer of class mu glutathione transferase from rat.

Although rat glutathione transferase M1-1 is crystallized as a homodimer (GST M1-1), we have generated monomers (GST M1) of the enzyme by adding potassium bromide to buffer solutions containing the wild-type enzyme and by introducing point mutations in the electrostatic region of the subunit interface. The wild-type enzyme was evaluated in 0.05 M MES (pH 6.5) containing up to 3 M KBr. We report that the addition of KBr greatly influences the monomer-dimer equilibrium of the wild-type enzyme and that at 3 M KBr GST M1 has a specific activity close to that of GST M1-1. Since the effect of KBr is likely due to charge screening at the subunit interface, the influence on the monomer-dimer equilibrium exerted by the amino acid residues in the electrostatic region of the interface (Arg77, Asp97, Glu100, Asn101) was investigated. Mutations introduced at positions 97, 100, and 101 promote monomerization, resulting in enzymes that exhibit a decreased weight average molecular weight in comparison to that of the wild-type enzyme. However, only mutations at position 97 result in enzymes that have catalytic activity in the monomeric form. The mutations introduced at positions 100 or 101 result in enzymes whose activity can be accounted for by the amount of dimeric enzyme present. Our results indicate that the electrostatic region of the interface is important in the monomer-dimer equilibrium of glutathione transferase and that, although GST M1-1 may be more active than GST M1, the dimer is not required for catalytic function.