Mechanism-Based Inactivation of Cytochrome P450 2B1 by 9-Ethynylphenanthrene

• Published in: Archives of biochemistry and biophysics 1995-11, Vol.323 (2), p.295-302
• Main Authors: Roberts, Elizabeth S., Eddy Hopkins, Nancy, Zaluzec, Eugene J., Gage, Douglas A., Alworth, William L., Hollenberg, Paul F.
• Format: Article
• Language: English
• Subjects: 7-Alkoxycoumarin O-Dealkylase - antagonists & inhibitors; 9-ethynylphenanthrene; Amino Acid Sequence; Animals; aryl acetylene; Aryl Hydrocarbon Hydroxylases; cytochrome; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - chemistry; Enzyme Inhibitors - chemistry; Kinetics; Male; mechanism-based; Microsomes, Liver - enzymology; Molecular Sequence Data; NADP - metabolism; P450 2B1; Peptide Mapping; Phenanthrenes - pharmacology; phenobarbital; Protein Binding; Rats; Steroid Hydroxylases - antagonists & inhibitors; Steroid Hydroxylases - chemistry
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1995.9961

The 7-ethoxycoumarinO-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°C, the extrapolated maximal rate constant of inactivation (kinactivation) was 0.45 min−1and 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 (ap proximately 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.