Selective Inhibition of a Cytochrome P450 Enzyme in Wheat That Oxidizes Both the Natural Substrate Lauric Acid and the Synthetic Herbicide Diclofop

• Published in: Pesticide biochemistry and physiology Vol. 54; no. 3; pp. 161 - 171
• Main Authors: Helvig, Christian, Tardif, François J., Seyer, André, Powles, Stephen B., Mioskowski, Charles, Durst, Francis, Salaün, Jean-Pierre
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01-03-1996; Elsevier
• Subjects: ACIDE GRAS SATURE; ACIDOS GRASOS SATURADOS; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Biological and medical sciences; Chemical control; CITOCROMO P 450; CITOCROMOS; CYTOCHROME; CYTOCHROME P 450; DICLOFOP; ENZIMAS; ENZYME; Fundamental and applied biological sciences. Psychology; INHIBICION; INHIBITION; ORGANITE CELLULAIRE; ORGANULOS CITOPLASMICOS; OXIDACION; OXYDATION; Parasitic plants. Weeds; Phytopathology. Animal pests. Plant and forest protection; PLANTULAS; PLANTULE; TRITICUM AESTIVUM; Weeds; Agricultural chemical product; Monocotyledones; Aralkanoic acid; Lauric acid; Biochemistry; Selectivity; Microsome; Cereal crop; Enzymatic activity; Gramineae; Angiospermae; Reaction mechanism; Triple bond; Oxidation; Phytopharmacology; Synthetic fatty acid; Hydroxylation; Cytochrome P450; Pesticides; Enzyme inhibitor; Substrate inhibition; Metabolism; Substrate analog; In vitro; Herbicide; Propionic acid(2-aryloxy); In vivo; Biochemical reaction; Spermatophyta; Triticum aestivum
• ISSN: 0048-3575; 1095-9939
• DOI: 10.1006/pest.1996.0020

Earlier studies from our laboratory strongly suggested that a single or similar cytochrome P450 isoform(s) isolated from microsomes of wheat catalyze the oxidation of the medium-chain fatty acid, lauric acid, and the wheat selective herbicide diclofop. Lauric acid is mainly hydroxylated at the subterminal position C11 (ω-1) and for that reason P450-dependent reactions were initially designated lauric acid (ω-1)-hydroxylase ((ω-1)-LAH). This report presents data on thein vitroandin vivoirreversible inhibition of both lauric acid and diclofop oxidation by mechanism-based inhibitors targeting lauric acid hydroxylation. Incubation of microsomes from etiolated wheat seedlings with 10-dodecynoic acid (10-DDYA) produces a dramatic inhibition of lauric acid hydroxylation. The inhibition is both time- and concentration-dependent in a process typical for mechanism-based inhibitors. A half-life of 3 min and an apparent inhibition constant of 14 μMwere determined from pseudo-first order kinetic studies of (ω-1)-LAH inhibition. Similar results were obtained by incubating microsomes with a terminal acetylene, 11-dodecynoic acid (11-DDYA). Based on results ofin vitroexperiments we have developed a series of new mechanism-based inhibitors to inhibit diclofop metabolism in developing wheat seedlings. To protect the inhibitors from degradation by α- and β-oxidation systems, water-soluble sodium salts of undec-10-yne-1-sulfonic acid (10-UDYS), undec-9-yne-1-sulfonic acid (9-UDYS), and undecan-1-sulfonic acid (SULAUR) were synthesized. Following treatment of wheat coleoptiles for 6 hr with these modified inhibitors, diclofop and chlortoluron metabolism was measured. Both compounds selectively inhibited diclofop metabolism without affecting chlortoluron metabolism. The importance of triple bonds in the inhibition process is clearly demonstrated by the fact that SULAUR had very small inhibitory effects. A 100 μMconcentration of both inhibitors resulted in about 50% inhibition of diclofop metabolism. Increasing the concentration of inhibitors to 2.5 mMincreased inhibition of diclofop oxidation to about 90%. Our results clearly show that one or similar forms of cytochrome P450 that are naturally involved in lauric acid oxidation are responsible for diclofop hydroxylation.