TY - JOUR
T1 - Suicide inactivation of cytochrome p450 by midchain and terminal acetylenes
T2 - A mechanistic study of inactivation of a plant lauric acid ω- hydroxlyase
AU - Helvig, Christian
AU - Alayrac, Carole
AU - Mioskowski, Charles
AU - Koop, Dennis
AU - Poullain, Didier
AU - Durst, Francis
AU - Salaün, Jean Pierre
PY - 1997
Y1 - 1997
N2 - Incubation of Vicia sativa microsomes, containing cytochrome P450- dependent lauric acid ω-hydroxylase (ω-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 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.
AB - Incubation of Vicia sativa microsomes, containing cytochrome P450- dependent lauric acid ω-hydroxylase (ω-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 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.
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U2 - 10.1074/jbc.272.1.414
DO - 10.1074/jbc.272.1.414
M3 - Article
C2 - 8995277
AN - SCOPUS:0031024928
SN - 0021-9258
VL - 272
SP - 414
EP - 421
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -