Identification of the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital.
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.
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Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.
Falick
A M
AM
Chen
B
B
Nilsson
F
F
Correia
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D019362:Cytochrome P-450 CYP2B1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D004586:Electrophoresis; D006418:Heme / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D051381:Rats; D017207:Rats, Sprague-Dawley; D012631:Secobarbital / Q000633:toxicity*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors
D065607:Cytochrome P-450 Enzyme Inhibitors; D010446:Peptide Fragments; D012631:Secobarbital; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C022724:testosterone 7-alpha-hydroxylase, hamster