Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine, and 1,2-dichloro-4-nitrobenzene

Protein Sci. 1999 Dec;8(12):2742-50. doi: 10.1110/ps.8.12.2742.


Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity (84%) exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG), and 1,2-dichloro-4-nitrobenzene (DCNB), hGST M2-2 being more efficient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the alpha6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg165-Glu164(-Gln167) was identified based on the published three-dimensional structure of hGST M2-2. Corresponding variant residues of hGSTM1-1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2-2 residues to the framework of hGST M1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequences were analyzed. The activity with 1-chloro-2,4-dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2-2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg165 in hGST M2-2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2-2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild-type hGST M2-2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2-2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Glutathione Transferase / chemistry*
  • Glutathione Transferase / genetics
  • Humans
  • Indolequinones*
  • Indoles / chemistry*
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nitrobenzenes / chemistry*
  • Nitrosoguanidines / chemistry*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Rats
  • Sequence Alignment
  • Substrate Specificity


  • Indolequinones
  • Indoles
  • Nitrobenzenes
  • Nitrosoguanidines
  • aminochrome 1
  • 1,3-dimethyl-2-cyano-1-nitrosoguanidine
  • 1,2-dichloro-4-nitrobenzene
  • Glutathione Transferase