Suicide inactivation of cytochrome p450 by midchain and terminal acetylenes: A mechanistic study of inactivation of a plant lauric acid ω- hydroxlyase

Christian Helvig, Carole Alayrac, Charles Mioskowski, Dennis Koop, Didier Poullain, Francis Durst, Jean Pierre Salaün

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)414-421
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number1
DOIs
StatePublished - 1997

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lauric acid
Alkynes
Cytochrome P-450 Enzyme System
Suicide
Cytochrome P-450 CYP4A
Metabolites
Proteins
Vicia sativa
Nucleophiles
Oxygenation
Mercaptoethanol
Alkylation
Microsomes
11-dodecynoic acid
Electrophoresis
Liver
Labeling
Labels
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Suicide inactivation of cytochrome p450 by midchain and terminal acetylenes : A mechanistic study of inactivation of a plant lauric acid ω- hydroxlyase. / Helvig, Christian; Alayrac, Carole; Mioskowski, Charles; Koop, Dennis; Poullain, Didier; Durst, Francis; Salaün, Jean Pierre.

In: Journal of Biological Chemistry, Vol. 272, No. 1, 1997, p. 414-421.

Research output: Contribution to journalArticle

Helvig, Christian ; Alayrac, Carole ; Mioskowski, Charles ; Koop, Dennis ; Poullain, Didier ; Durst, Francis ; Salaün, Jean Pierre. / Suicide inactivation of cytochrome p450 by midchain and terminal acetylenes : A mechanistic study of inactivation of a plant lauric acid ω- hydroxlyase. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 1. pp. 414-421.
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abstract = "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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