IDENTIFICATION OF THE HEME ADDUCT AND AN ACTIVE-SITE PEPTIDE MODIFIEDDURING MECHANISM-BASED INACTIVATION OF RAT-LIVER CYTOCHROME-P450 2B1 BY SECOBARBITAL

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic know n to be a relatively selective mechanism-based inactivator of rat live r cytochrome P450 2B1. Previous studies have demonstrated that such in activation results in prosthetic heme destruction and irreversible dru g-induced protein modification, events most likely triggered by P450 2 B1-dependent oxidative activation of the olefinic pi-bond. However, th e precise structure of the SE-modified heme and/or the protein site ta rgeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [C-14]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 (MALD I-TOF), and liquid secondary ion mass spectrometry in the positive mod e (+LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric a cid)protoporphyrin IX adduct, The [C-14]SB-modified 2B1 protein has al so been isolated from similar inactivation systems and subjected to ly syl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [C-1 4]SB-modified 2B1 peptide was thus isolated, purified, electrotransfer red onto a poly(vinylidene) membrane, and identified by micro Edman de gradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISL L(293)) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the dis tal 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 intim ately contact the substrates. This finding thus suggests that SB-induc ed protein modification of P450 2B1 also occurs at the active site and , together with heme N-alkylation, contributes to the SE-induced mecha nism-based inactivation of P450 2B1.