title: Role of the highly conserved threonine in cytochrome P450 2E1: prevention of H2O2-induced inactivation during electron transfer.
abstract: A highly conserved threonine in the I-helix of cytochrome P450s has been suggested to play an important role in dioxygen activation, a critical step for catalytic turnover. However, subsequent studies with some P450s in which this highly conserved threonine was replaced by another residue such as alanine showed that significant catalytic activities were still retained when the variants were compared with the wild type enzymes. These results make the role of this residue unclear. We provide data here that suggest a novel role for this highly conserved threonine (Thr303) in the function of P450 2E1. We found that the P450 2E1 T303A mutant undergoes rapid autoinactivation in the reconstituted system during catalytic turnover when the electrons are provided by NADPH. This inactivation was much faster than that of the wild type P450 2E1 and was prevented by catalase. Both the P450 2E1 wild type and T303A mutants produce hydrogen peroxide during the incubations. The inactivation was accompanied by heme destruction with part of the heme becoming covalently attached to protein. The heme destruction was prevented by catalase or by the presence of substrate. Interestingly, this inactivation occurred much more rapidly in the presence of both an electron transfer system and hydrogen peroxide externally added to the enzyme. This accelerated inactivation during catalytic turnover was also found with a 2B4 T302A mutant, which corresponds to 2E1 T303A. Our results suggest that the conserved threonine in these P450s prevents rapid autoinactivation during the catalytic cycle and that this residue may be highly conserved in P450s since it allows them to remain catalytically active for longer periods of time.
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School , 2301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-5632, United States.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Yoshigae
authors.forename: Ute M
authors.initials: UM
authors.lastname: Kent
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D000975:Antioxidants / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000235:genetics; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009154:Mutation; D009249:NADP / Q000737:chemistry
chemical_list: D000975:Antioxidants; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D006418:Heme; D009249:NADP; D006861:Hydrogen Peroxide; D019392:Cytochrome P-450 CYP2E1