Identification of the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital

Chem Res Toxicol. Apr-May 1996;9(3):614-22. doi: 10.1021/tx950177k.

Abstract

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic known to be a relatively selective mechanism-based inactivator of rat liver cytochrome P450 2B1. Previous studies have demonstrated that such inactivation results in prosthetic heme destruction and irreversible drug-induced protein modification, events most likely triggered by P450 2B1-dependent oxidative activation of the olefinic pi-bond. However, the precise structure of the SB-modified heme and/or the protein site targeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [14C]SB in a functionally reconstituted system and structurally characterized it by electronic absorption spectroscopy and tandem collision-induced dissociation (CID), matrix-assisted laser desorption ionization on time of flight (MALDI-TOF), and liquid secondary ion mass spectrometry in the positive mode (+ LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX adduct. The [14C]SB-modified 2B1 protein has also been isolated from similar inactivation systems and subjected to lysyl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [14C]SB-modified 2B1 peptide was thus isolated, purified, electrotransferred onto a poly-(vinylidene) membrane, and identified by micro Edman degradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISLL293) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the distal helix I of P450 101, a region highly conserved through evolution, and which is known not only to flank the heme moiety but also to intimately contact the substrates. This finding thus suggests that SB-induced protein modification of P450 2B1 also occurs at the active site and, together with heme N-alkylation, contributes to the SB-induced mechanism-based inactivation of P450 2B1.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Binding Sites
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 CYP2B1
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / isolation & purification
  • Electrophoresis
  • Heme / analysis*
  • Heme / chemistry
  • Heme / metabolism
  • Male
  • Mass Spectrometry
  • Microsomes, Liver / drug effects*
  • Microsomes, Liver / metabolism
  • Molecular Sequence Data
  • Oxidoreductases / antagonists & inhibitors
  • Peptide Fragments / chemistry
  • Peptide Mapping
  • Rats
  • Rats, Sprague-Dawley
  • Secobarbital / toxicity*
  • Steroid Hydroxylases / antagonists & inhibitors

Substances

  • Cytochrome P-450 Enzyme Inhibitors
  • Peptide Fragments
  • Secobarbital
  • Heme
  • Cytochrome P-450 Enzyme System
  • Oxidoreductases
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP2B1
  • testosterone 7-alpha-hydroxylase, hamster