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

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 wi th the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosogunnidine (cya noDMNG), and 1,2-dichloro-4-nitrobenzene (DCNB), hGST M2-2 being more effic ient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also s howing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the alpha 6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mou se Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg-165-Gl u164(-Gln167) was identified based on the published three-dimensional struc ture of hGST M2-2. Corresponding variant residues of hGSTM1-1 (Leu165. Asp1 64. 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 M 1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequence s were analyzed. The activity with 1-chloro-2,4-dinitrobenzene (CDhTB) was retained in all mutant enzymes, proving that they were catalytically compet ent, 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 Arg1 65 in hGST M2-2 appears to be important for the reactions with cyanoDMNG an d DCNB. A mutant, which contained all the hGST M2-2 residues of the putativ e electrostatic network, was still lacking one order of magnitude of the ac tivities 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 cha racteristic activities with aminochrome, cyanoDMNG, and DCNB.