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Glutathione (GSH) transferases (GSTs) play an important role in detoxification of (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated metabolite of widespread environmental pollutant benzo[a]pyrene (BP). Accumulating evidence indicates that Alpha class human GST isoforms (hGSTA) may play an important role in defense against DNA damaging effect of (+)-anti-BPDE in some tissues (e.g., liver and lung) because of their high abundance. Crystal structure of murine mGSTA1-1 in complex with GSH conjugate of (+)-anti-BPDE suggested that a salt-bridge between amino acid residues Arg-220 and Asp-41 might be responsible for significantly lower activity of hGSTA1-1 towards (+)-anti-BPDE in comparison with mGSTA1-1. In the present study, we experimentally tested this possibility by determining the effects of Arg-220-Ala and Asp-41-Ala mutations, singly as well as in combination, on GSH-(+)-anti-BPDE conjugating activity of hGSTA1-1. Mutation of Arg-220-Ala and/or Asp-41-Ala did not have any appreciable effect on catalytic efficiency of hGSTA1-1 towards (+)-anti-BPDE. These results argue against a role of Arg-220/Asp-41 salt-bridge in hGSTA1-1 catalyzed GSH conjugation of (+)-anti-BPDE. hGSTA1-1 shares about 95% amino acid sequence similarity with hGSTA2-2, yet the former isoform is >5-fold more efficient catalyst of (+)-anti-BPDE-GSH conjugation than hGSTA2-2. The amino acid sequence alignment of hGSTA1-1 and hGSTA2-2 reveals 10 substitutions but only 4 of these changes are located in the hydrophobic substrate binding site (residues in positions 9, 11, 110 and 215). Mutation of Ile-11 of hGSTA2-2 to Ala (which is present in the same position in hGSTA1-1) resulted in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution was sufficient to convert hGSTA2-2 to a protein matching hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/Ile-11-Ala was accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (Ser-9-Phe, Ile-11-Ala, Phe-110-Val and Ser-215-Ala mutations) resulted in an enzyme whose catalytic efficiency was 13-fold higher than that of the wild type hGSTA2-2. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. In conclusion, the results of the present study offer insights into the structural basis for catalytic differences between closely related hGSTA1-1 and hGSTA2-2 for GSH conjugation of (+)-anti-BPDE (supported by NCI grant CA076348 and NIEHS grant ES09140).

[Proc Amer Assoc Cancer Res, Volume 46, 2005]