An alpha class mouse glutathione S-transferase with exceptional catalytic efficiency in the conjugation of glutathione with 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene

Abstract

The kinetics of the conjugation of glutathione (GSH) with anti-7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene (anti-BPDE) catalyzed by GSH S-transferase (GST) isoenzymes purified from the liver and forestomach of female A/J mouse has been investigated. The GST isoenzymes studied included an alpha class isoenzyme of forestomach (GST 9.5), alpha class hepatic isoenzymes mGSTA3-3 and mGSTA4-4, pi class hepatic isoenzyme mGSTP1-1, and mu class hepatic isoenzyme mGSTM1-1. When the concentration of (+)-anti-BPDE was varied (5-120 microM) at a fixed GSH concentration (2 mM), linear Lineweaver-Burk plots were observed for each isoenzyme. The kcat values for GST 9.5, mGSTA3-3, mGSTP1-1, mGSTM1-1, and mGSTA4-4 were 2.0, 0.02, 0.40, 0. 05, and 0.01 s-1, respectively, with corresponding Km values of 16, 12, 29, 27, and 49 microM. The catalytic efficiency (kcat/Km) of GST 9.5 in the conjugation of GSH with (+)-anti-BPDE, which is believed to be the ultimate carcinogenic metabolite of benzo(a)pyrene, was about 9-625-fold higher as compared with other mouse GST isoenzymes. These results indicate that GST 9.5 of forestomach is different among mammalian alpha class GSTs because (+)-anti-BPDE has been shown to be a poor substrate for alpha class rat or human GST isoenzymes. The catalytic efficiency of GST 9.5 was approximately 4.5-fold higher than that of pi class human isoenzyme (hGSTP1-1), which among human GSTs is reported to be most efficient in the detoxification of (+)-anti-BPDE. Unlike rat GST isoenzymes, linear Lineweaver-Burk plots were observed for mouse GSTs when GSH was used as a variable substrate. The catalytic efficiencies of the mouse GSTs toward (+)-anti-BPDE were about 2-20-fold higher as compared with the (-)-enantiomer of anti-BPDE. The results of the present study suggest that GST 9.5 may play an important role in the detoxification of (+)-anti-BPDE.