SUICIDE INACTIVATION OF CYTOCHROME-P450 BY MIDCHAIN AND TERMINAL ACETYLENES - A MECHANISTIC STUDY OF INACTIVATION OF A PLANT LAURIC ACID OMEGA-HYDROXLYASE

Incubation of Vicia sativa microsomes, containing cytochrome P450-depe ndent lauric acid omega-hydroxylase (omega-LAH), with [1-C-14]11-dodec ynoic acid (11-DDYA) generates a major metabolite characterized as 1,1 2-dodecandioic acid, In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible bindin g 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 associat ed with several protein bands of about 53 kDa, The presence of beta-me rcaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxi dized 11-DDYA with the nucleophile, Although the alkylation of protein s was reduced, the lauric acid omega-hydroxylase activity was not rest ored, suggesting an active site-directed inactivation mechanism, Simil ar results were obtained when reconstituted mixtures of cytochrome P45 0 from family CYP4A from rabbit liver were incubated With 11-DDYA, In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cy tochrome P450 2B4 protein and irreversible inhibition of activity, The se results demonstrate that acetylenic analogues of substrate are effi cient mechanism based inhibitors and that a correlation between the po sition of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.