Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid -hydroxylase (-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 concentrationdependent 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

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... alysis 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 mechanismbased 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. Supported by a BDI fellowship from Rhône-Poulenc. 1 The abbreviations used are: -LAH, -hydroxylase of lauric acid; CYP, cytochrome P; DDYA, dodecynoic acid; diacid, 1,12-dodecandioic acid; GC-MS, gas chromatography-mass spectrometry; LMW, low molecular weight; MeTMS, trimethylsylil ether methyl ester; RP-HPLC, reverse phase high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis. 2 N. Tijet and I. Benveniste, unpublished results. 3 I. Benveniste, unpublished results.