DISTAL SITE AND SURFACE MUTATIONS OF CYTOCHROME-P450 1A2 MARKEDLY ENHANCE DEHALOGENATION OF CHLORINATED HYDROCARBONS

Chlorinated compounds such as chlorinated ethylenes and ethanes are se rious environmental pollutants, In the present study, we examined whet her or not a recombinant strain of Saccharomyces cerevisiae that expre sses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant pro teins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane, Mutati ons at putative heme distal and protein surface sites of P450 1A2 grea tly enhanced turnover values toward those substrates under both aerobi c and anaerobic conditions, For example, a Thr319Ala mutation at the p utative heme distal site enhanced the degradation rate of trichloroeth ylene and pentachloroethane by 2- and 2.7-fold, respectively, under ae robic conditions, The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic a nd anaerobic conditions, respectively, The Thr319Ala mutation increase d dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate, A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobi c conditions, In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pen tachloroethane substrates under aerobic conditions, Conserved amino ac ids Thr319 and Glu318 at the heme distal site have been suggested to b e important in the O-2 activation during monooxidation reactions of P4 50s, However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta-and hexa chloroethanes, The roles of Thr319, Glu318, and Lys250 in the catalysi s with chlorinated hydrocarbons are discussed in association with reac tion mechanisms. (C) 1997 Academic Press.