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Volume 272, Number 1, Issue of January 3, 1997 pp. 414-421
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Suicide Inactivation of Cytochrome P450 by Midchain and Terminal Acetylenes
A MECHANISTIC STUDY OF INACTIVATION OF A PLANT LAURIC ACID -HYDROXLYASE

(Received for publication, May 25, 1996, and in revised form, October 9, 1996)

Christian Helvig , Carole Alayrac ^¶ , Charles Mioskowski ^¶ , Dennis Koop ^** , Didier Poullain , Francis Durst and Jean-Pierre Salaün

From the  Institut de Biologie Moléculaire des Plantes-CNRS, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France, ^¶ CNRS-URA 1386 Faculté de Pharmacie, Laboratoire de Synthèse Bio-Organique, 74 route du Rhin, F-67048 Strasbourg Cedex, France, ^** Department of Pharmacology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3098, and  Commissariat à l'Energie Atomique, Service des Molécules Marquées, F-91191 Gif-sur-Yvette Cedex, France

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid -hydroxylase (-LAH), with [1-¹⁴C]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 -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 -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.

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