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.
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.
Hansson
L O
LO
Department of Biochemistry, Uppsala University, Biomedical Center, Sweden.
Bolton-Grob
R
R
Widersten
M
M
Mannervik
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D045563:Indolequinones; D007211:Indoles / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009578:Nitrobenzenes / Q000737:chemistry*; D009604:Nitrosoguanidines / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity
D045563:Indolequinones; D007211:Indoles; D009578:Nitrobenzenes; D009604:Nitrosoguanidines; C091584:aminochrome 1; C063048:1,3-dimethyl-2-cyano-1-nitrosoguanidine; C028328:1,2-dichloro-4-nitrobenzene; D005982:Glutathione Transferase