Suicide Inactivation of Cytochrome P450 by Midchain and Terminal Acetylenes

• Published in: The Journal of biological chemistry Vol. 272; no. 1; pp. 414 - 421
• Main Authors: Helvig, Christian, Alayrac, Carole, Mioskowski, Charles, Koop, Dennis, Poullain, Didier, Durst, Francis, Salaün, Jean-Pierre
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 03.01.1997
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.1.414

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid ω-hydroxylase (ω-LAH), with [1- 14 C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of β-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid ω-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.