title: Mechanism-based inactivation of cytochrome P450 2B1 by 9-ethynylphenanthrene.
abstract: The 7-ethoxycoumarin O-deethylase activity of rat cytochrome P450 (P450) 2B1 was inactivated by 9-ethynylphenanthrene (9EPh) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. At 20 degrees C, the extrapolated maximal rate constant of inactivation (kinactivation) was 0.45 min-1 and the inactivator concentration required for half-maximal inactivation (KI) was 138 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis demonstrated that [2'-3H]-9EPh was irreversibly bound to the protein moiety of P450 2B1 and the stoichiometry of binding was determined to be 0.82 mol of inactivator bound per mole of P450 2B1. A radiolabeled peptide of approximately 3.0 kDa was identified by autoradiography after Tricine SDS-PAGE analysis of the peptides generated from a cyanogen bromide cleavage of [2'-3H]9EPh-inactivated P450 2B1. After HPLC separation of these peptides, the fraction containing the most radioactivity was analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and peaks at m/z 2720.9 and 2939.9 were detected. The lower mass peak represents the molecular ion (MH+) for the peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the MH+ of the modified peptide (theoretical 2940.5). The difference in mass (approximately 219) would correspond to the addition of a phenanthrylacetyl group to the peptide. When the fraction containing the modified and unmodified peptides was further digested with pepsin and reanalyzed by MALDI-MS, the site of attachment could be assigned to one of the amino acids contained in the peptide Phe297 to Leu307.
mesh_terms: D015660:7-Alkoxycoumarin O-Dealkylase / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010449:Peptide Mapping; D010616:Phenanthrenes / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: C096666:9-ethynylphenanthrene; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D010616:Phenanthrenes; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D015660:7-Alkoxycoumarin O-Dealkylase; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase