Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase.
Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-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 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 beta-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 omega-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.
Helvig
C
C
Institut de Biologie Moléculaire des Plantes-CNRS, Département d'Enzymologie Cellulaire et Moléculaire, Strasbourg, France. helvigC@ctrvax.vanderbilt.edu
Alayrac
C
C
Mioskowski
C
C
Koop
D
D
Poullain
D
D
Durst
F
F
Salaün
J P
JP
D000114:Acetylene / Q000737:chemistry; D000818:Animals; D042926:Cytochrome P-450 CYP4A; D065607:Cytochrome P-450 Enzyme Inhibitors; D006418:Heme / Q000737:chemistry; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D010940:Plant Proteins / Q000037:antagonists & inhibitors; D010944:Plants; D011817:Rabbits; D013329:Structure-Activity Relationship
D065607:Cytochrome P-450 Enzyme Inhibitors; D007850:Lauric Acids; D010940:Plant Proteins; C030358:lauric acid; D006418:Heme; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D000114:Acetylene