Autocatalytic inactivation of plant cytochrome P-450 enzymes: Selective inactivation of the lauric acid in-chain hydroxylase from Helianthus tuberosus L. by unsaturated substrate analogs

doi:10.1016/0003-9861(84)90515-0

Archives of Biochemistry and Biophysics

Volume 232, Issue 1, July 1984, Pages 1-7

https://doi.org/10.1016/0003-9861(84)90515-0 Get rights and content

Abstract

Lauric acid in-chain hydroxylation is inhibited in microsomes from Jerusalem artichoke tubers (Helianthus tuberosus L.) incubated with 9-decenoic, 11-dodecenoic, or 11-dodecynoic acids. 9-Decenoic acid is at best a weak competitive inhibitor of the inchain hydroxylase, but inactivates the enzyme in a time-dependent, pseudo-first-order process with a rate constant of approximately 1.1 × 10⁻³ s⁻¹. In contrast, 11-dodecenoic acid causes a slower, time-dependent loss of the hydroxylase activity, but is a potent competitive inhibitor of the enzyme (K_i = 2 μM). Neither agent decreases the microsomal concentration of cytochrome b₅, NADH-cytochrome b₅ reductase, or NADPH cytochrome P-450 reductase. Cinnamic acid 4-hydroxylation, catalyzed by a cytochrome P-450 enzyme, is not affected by concentrations of 9-decenoic acid that suppress lauric acid hydroxylation. 11-Dodecenoic acid is much less specific and, at higher concentrations, markedly reduces the microsomal cytochrome P-450 content, and the hydroxylation of both lauric and cinnamic acids.

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Cited by (14)

Carboxylic submetabolome-driven signature characterization of COVID-19 asymptomatic infection
2022, Talanta
Citation Excerpt :
9-decenoic acid was regarded as the main contributors of β-oxidation [20]. Salaun et al. found that 9-decenoic acid was a weak competitive inhibitor of the in-chain hydroxylase, which can inactivate the P450 enzyme [26]. we hypothesized 9-decenoic acid played a similar role in asymptomatic patients.
Asymptomatic infection of COVID-19 is a global threat for public health. Unfortunately, the study about metabolic dysregulation of asymptomatic infection is barely investigated. Here, we performed carboxylic submetabolome profiling of serum from 62 asymptomatic and 122 control individuals, by a highly sensitive chemical isotope labelling method. Twenty-one discriminative carboxylic features, including 12-hydroxyeicosatetraenoic acid, cholic acid, glycoursodeoxycholic acid and 15,16-dihydroxyoctadeca-9,12-dienoic acid were discovered to be dysregulated in asymptomatic patients. This panel containing 21 carboxylic features could accurately identify asymptomatic patients based on a random forest model, providing an accuracy of 85.7% with only 3.6% false positive rate and 7.1% false negative rate. The dysregulated metabolites found in asymptomatic patients covered several important pathways, such as arachidonic acid metabolism, synthesis of bile acid, β-oxidation of fatty acids, activation of macrophage and platelet aggregation. This work provided valuable knowledge about serum biomarkers and molecular clues associated with asymptomatic COVID-19 patients.
Suicide inactivation of cytochrome p450 by midchain and terminal acetylenes: A mechanistic study of inactivation of a plant lauric acid ω- hydroxlyase
1997, Journal of Biological Chemistry
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.
Selective inhibition of a cytochrome P450 enzyme in wheat that oxidizes both the natural substrate laurie acid and the synthetic herbicide diclofop
1996, Pesticide Biochemistry and Physiology
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.
A novel imidazole carboxylic acid ester is a herbicide inhibiting 14α-methyl-demethylation in plant sterol biosynthesis
1991, Pesticide Biochemistry and Physiology
CGA 201029/R 69020 [methyl 1-(2,2-dimethylindan-1-yl)imidazole-5-carboxylate], a novel imidazole carbonic acid ester, inhibits sterol biosynthesis in plants. Changes in sterol pattern were observed in maize roots exposed to 10⁻⁸ M of the inhibitor for 7 days. The compound lowered contents of stigma-, campe-, and sitosterol, while obtusifoliol and 14α-methylergast-8-en-3β-ol accumulated. Label from [¹⁴C]mevalonic acid mainly accumulated in obtusifoliol when tobacco cell suspension cultures were exposed to CGA 201029/R 69020, while label was predominantly found in 4-demethylsterols of controls. The compound interacted with a microsomal cytochrome P450 as shown by binding spectra studies. These data suggest that this substance inhibits obtusifoliol 14-methyl demethylase in plants.
Epoxidation of cis and trans Δ<sup>9</sup>-unsaturated lauric acids by a cytochrome P-450-dependent system from higher plant microsomes
1989, FEBS Letters
A new epoxygenase, differing in substrate and stereospecificity from those previously described in mammals, bacteria and Vicia faba, has been characterized in higher plants. A microsomal fraction from Jerusalem artichoke tubers which catalyses in-chain monohydroxylation of lauric acid has now been found to convert synthetic 9-dodecenoic acid (9-DDNA) to 9,10-epoxylauric acid. Epoxidation of the cis and trans isomers proceeds at similar rates affording the corresponding cis and trans epoxides. This reaction is dependent upon O₂ and NADPH and does not occur with boiled microsomes. Lauric acid, carbon monoxide and polyclonal antibody against NADPH-cytochrome P-450 reductase from Jerusalem artichoke inhibit epoxide formation. Compared to freshly sliced tuber tissues, epoxidation activity was strongly enhanced in tissues aged in 20 mM aminopyrine solution. The possibility that 9, 10-epoxidation of 9-DDNA is catalyzed by the cytochrome P-450 isoenzyme involved in lauric acid in-chain hydroxylation in this plant was investigated.
Differential inactivation of plant lauric acid ω- and in-chain-hydroxylases by terminally unsaturated fatty acids
1988, Archives of Biochemistry and Biophysics
The microsomal fraction from Vicia sativa L. cv. Septimane contains a cytochrome P-450-dependent lauric acid ω-hydroxylase that is inactivated in a time-dependent, pseudo-first-order manner when the microsomes are incubated with 11-dodecynoic acid. The rate constant for the inactivation is approximately 4.3–4.8 × 10⁻³ s⁻¹. In contrast, the olefinic analog 11-dodecenoic acid is primarily a time-independent inhibitor of the ω-hydroxylase. 1-Aminobenzotriazole, 3-phenoxy-1-propyne, and 3-(2,4-dichlorophenoxy)-1-propyne, mechanism-based inactivators of cinnamic acid 4-hydroxylase, and 9-decenoic acid, a mechanism-based inactivator of the lauric acid in-chain hydroxylase, are at best poor inactivators of the ω-hydroxylase. Conversely, cinnamic acid 4-hydroxylase is only slightly affected by concentrations of 11-dodecynoic acid that completely inactivate the ω-hydroxylase. 11-Dodecynoic acid is thus a potent, relatively specific, inactivator of the V. sativa lauric acid ω-hydroxylase.

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^☆: This research was supported by ATP Research Grant 4117 (Strasbourg), National Institutes of Health Grant GM 25515 (San Francisco), and North Atlantic Treaty Organization Travel Grant RG 109.80.

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Autocatalytic inactivation of plant cytochrome P-450 enzymes: Selective inactivation of the lauric acid in-chain hydroxylase from Helianthus tuberosus L. by unsaturated substrate analogs☆

Abstract

Phytochemistry

Phytochemistry

Pharmacol. Ther

Biochim. Biophys. Acta

Tetrahedron

Arch. Biochem. Biophys

J. Biol. Chem

Phytochemistry

Eur. J. Biochem

Physiol. Veg

Amer. J. Bot

J. Liq. Chromatogr