Suicide Inactivation of Cytochrome P450 by Midchain and Terminal Acetylenes
A MECHANISTIC STUDY OF INACTIVATION OF A PLANT LAURIC ACID omega -HYDROXLYASE*

(Received for publication, May 25, 1996, and in revised form, October 9, 1996)

Christian Helvig Dagger §, Carole Alayrac par , Charles Mioskowski , Dennis Koop **, Didier Poullain Dagger Dagger , Francis Durst Dagger and Jean-Pierre Salaün Dagger

From the Dagger  Institut de Biologie Moléculaire des Plantes-CNRS, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France,  CNRS-URA 1386 Faculté de Pharmacie, Laboratoire de Synthèse Bio-Organique, 74 route du Rhin, F-67048 Strasbourg Cedex, France, ** Department of Pharmacology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3098, and Dagger Dagger  Commissariat à l'Energie Atomique, Service des Molécules Marquées, F-91191 Gif-sur-Yvette Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega -hydroxylase (omega -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 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.


INTRODUCTION

The biological roles of plant P450 fatty acid hydroxylases are poorly understood. Evidence suggests that long-chain fatty acid omega -hydroxylases such as the oleic acid omega -hydroxylase (1, 2) may play a role in plant cuticle biosynthesis. The terminal hydroxyl group appears to be required for the polymerization of cutin monomers. In view of the differential inhibition of omega -LAH1 and oleic acid omega -hydroxylase activities from Vicia sativa by mechanism-based inhibitors and aliphatic acyltriazole derivatives, it was suggested that both reactions are catalyzed by different forms of P450 in V. sativa seedlings (2).

The enzymatic and structural characterization of plant P450s has been hampered by the low specific contents of these enzymes and by the difficulties encountered during their solubilization and purification (3, 4). Unfortunately, our efforts to purify fatty acid hydroxylases were unsuccessful, and only partly purified preparations2 exhibiting omega -LAH activity after reconstitution have been obtained.

A remarkable property of the plant fatty acid hydroxylases is their inducibility by clofibrate (1, 5), a hypolipidemic drug that induces peroxisomal proliferation in mammals (6, 7) and plants (8).

Although several similarities exist between omega -hydroxylases from plants and mammals (i.e. selective induction by arylphenoxy compounds and phthalate esters, similar substrate specificity, and inactivation by terminal acetylene), antibodies raised against the rat liver lauric acid omega -hydroxylase (CYP4A1) neither inhibit omega -LAH activity from plants nor interact in Western blotting with microsomal proteins from V. sativa.3

Mechanism-based inhibitors containing a terminal acetylene were previously shown to be potent irreversible inhibitors of both plant (9, 10) and mammalian (11, 12) fatty acid omega -hydroxylases. Examples of in vitro and in vivo inactivation of mammalian fatty acid omega -hydroxylases by these compounds have been published (13, 14, 15). Inhibition of enzyme activity was usually associated with the inhibition-dependent alkylation of the enzyme heme group and the apoprotein. Thus one goal of the current work was to develop compounds that might selectively label plant fatty acid hydroxylases as an aid to monitor the purification of these proteins.

Fatty acid acetylenic analogues have been used successfully to explore the functional role and the reaction mechanisms of several mammalian fatty acid omega -hydroxylases (15, 16). With the goal of selectively inhibiting the plant omega -LAH, we developed a series of radiolabeled acetylenic analogues of lauric acid, [1-14C]8-, 9-, 10-, and 11-dodecynoic acids (DDYAs) (Fig. 1). We report here results of microsomal incubations from clofibrate-treated V. sativa seedlings, containing a lauric acid omega -hydroxylase, with this series of acetylenes. The mechanism by which the plant omega -LAH was inactivated by the radiolabeled acetylenic 11-dodecynoic acid was investigated, metabolites generated were identified, and the chemical labeling of microsomal proteins was demonstrated. For comparison, the effects of 10- and 11-DDYA on the oxidation of lauric acid by mammalian CYP4A and 2B4 were investigated.


Fig. 1. Structures of substrates and metabolites.
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MATERIALS AND METHODS

Chemicals

Radiolabeled [1-14C]lauric acid (45 Ci/mol) was from Commissariat à l'Energie Atomique (Gif-sur-Yvette, France). Thin-layer plates (Silica gel G60 F254) were from Merck (Darmstadt, Germany). Clofibrate and NADPH were purchased from Sigma. Dilauroylphosphatidylglycerol was from Calbiochem, and silylating reagent (N,O-bis-(trimethylsilyl)trifluoroacetamide + trimethylchlorosilane) was from Pearce Europe (Oud-Beijerland, The Netherlands).

Synthesis of Acetylenic Analogues

[1-14C]8-, 9-, 10-, and 11-dodecynoic acids were synthesized from 1-bromo-6-hexanol, 1-bromo-7-heptanol, and 1-bromo-9-nonanol by established procedures (17). [1-14C]Dodecynoic acids were prepared by Commissariat à l'Energie Atomique with a radiochemical purity greater than 98% and a specific activity of 42.3 Ci/mol.

Microsomal Preparation

V. sativa L. (var. Lolita) seedlings were purchased from S. A. Blondeau (Bersée, France). Four-day-old etiolated seedlings were aged for 72 h in 1 mM clofibrate solution before isolation of microsomal fractions as described by Salaün et al. (5). The 100,000 × g microsomal pellets were resuspended in either 50 mM phosphate buffer (pH 7.4), 30% (v/v) glycerol or in the same mixture containing 1.5 mM of beta -mercaptoethanol. Microsomal proteins were quantified by a microassay procedure from Bio-Rad using bovine serum albumin as a standard. P450 was measured by the method of Omura and Sato (18).

Enzyme Activity and Inhibition

Enzymatic activities were measured as described previously (19, 20). The standard assay contained in a final volume of 0.2 ml 0.19-0.43 mg of microsomal protein, 20 mM phosphate buffer (pH 7.4), and radiolabeled substrate. omega -Hydroxylase activities were measured in the presence of 1 mM NADPH plus a regenerating system (consisting of final concentration of 6.7 mM glucose-6-phosphate and 0.4 IU of glucose-6-phosphate dehydrogenase), and 375 µM beta -mercaptoethanol. The reaction was initiated with NADPH at 27 °C and stopped with 0.2 ml of acetonitrile/acetic acid (99.8:0.2, v/v), mixed, and aliquots (100 µl) were directly spotted onto silica plates or injected into a RP-HPLC system. For metabolite isolation and characterization, incubates were extracted twice with diethyl ether. The solvent was dried, and the residue was dissolved in methanol for TLC and RP-HPLC analysis. Products were quantified by liquid scintillation.

For carbon monoxide inhibition studies, the incubates were equilibrated with a stream of a CO/O2/N2 (2:2:8, v/v/v) mixture at 4 °C for 20 min. After equilibration at 27 °C for 5 min, reactions were initiated with NADPH and carried out either in darkness or under a white light from a 150-watt heat-filtered quartz lamp located 15 cm from the sample.

Monoclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke tubers (21) were preincubated with microsomes at 20 °C for 15 min before enzyme activity measurement as described previously (1).

Inactivation with Mechanism-based Inhibitors

The procedure of Salaün and co-workers (10) was followed. Microsomes were preincubated at 27 °C with 1 mM NADPH plus a regenerating system in the presence of variable concentrations of [1-14C]11-DDYA. At different times, 100 µl of preincubated microsomes was added to the incubation medium, which contained 100 µM [1-14C]lauric acid, 1 mM NADPH, and the regenerating system in a total volume of 200 µl. Incubations were performed for 5 min at 27 °C and stopped as described above.

Chromatographic Analysis

Metabolites were resolved by silica thin-layer chromatography with a mixture of diethyl ether/light petroleum (boiling point, 40-60 °C)/formic acid (70:30:1, v/v/v). The areas corresponding to 12-hydroxylauric acid (RF = 0.45) and 1,12-dodecandioic acid (RF = 0.6) were scraped directly into counting vials or eluted with ether and subjected to RP-HPLC analysis using a mixture of water/acetonitrile/acetic acid (25:75:0.2) as described previously (20, 22).

Radioactivity of RP-HPLC effluents was monitored with a computerized on-line solid scintillation counter (Ramona-D; Raytest, Germany).

Chemical Hydrogenation of Metabolites

Compounds isolated by RP-HPLC were chemically hydrogenated (H2 and Pd/charcoal) as described by Weissbart et al. (19) and subjected again to RP-HPLC analysis before mass spectra analysis.

Gas Chromatography-Mass Spectrometry

Gas chromatography and electron impact (70 eV) ionization mass spectrometry studies were performed as described elsewhere (19). The mass spectrum of the methyl ester derivative of the enzymatic product formed from [1-14C]11-DDYA showed predominant ions at m/z 98 (base peak), m/z 227 (M-31, 28%), m/z 185 (M-73, 30%), and m/z 153 (32%). This spectrum is similar to that obtained with the methyl ester of authentic 1,12-dodecandioic acid.

Metabolites generated from [1-14C]8-, 9-, and 10-DDYA by the enzyme were hydrogenated, methylated, and silylated as described by Weissbart et al. (19) and subjected to GC-MS analysis. The mass spectrum of the major metabolite generated from 10-DDYA (Fig. 2C, peak 5) was similar to that obtained with the authentic 12-hydroxylaurate methyl ester trimethylsilyl ether derivative (MeTMS). Similar mass spectra were obtained for metabolites generated from 8- and 9-DDYA (Fig. 2, A, peak 1, and B, peak 3).


Fig. 2. Representative TLC chromatograms of 8-, 9-, and 10-DDYA metabolites generated by microsomes from clofibrate-treated V. sativa seedlings. Microsomal fractions were incubated for 20 min with [1-14C]8-dodecynoic acid (A), [1-14C]9-dodecynoic acid (B), or [1-14C]10-dodecynoic acid (C) in the presence of NADPH at 27 °C. Aliquots (100 µl) of the incubated medium containing 50% of acidified acetonitrile were spotted on TLC layers. Radiolabeled compounds were resolved into 12-hydroxy-8-dodecynoic acid (peak 1), 12-hydroxy-9-dodecynoic acid (peak 3), 12-hydroxy10-dodecynoic acid (peak 5), residual 8-dodecynoic acid (peak 6), 9-dodecynoic acid (peak 7), and 10-dodecynoic acid (peak 8). Peaks 2 and 4, Unknown metabolites.
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Reconstitution of Activity from Partly Purified P450 Preparation

A partially purified mixture of CYP4A5, -4A6, and -4A7 (CYP4As) was obtained from rabbit kidney during the preparation of P4502CAA (23). CYP2B4 was isolated as described by Coon et al. (24), and purified rabbit NADPH cytochrome P450 reductase and rabbit cytochrome b5 were obtained as described by Laethem et al. (23). A purified NADPH cytochrome P450 reductase from Helianthus tuberosus (21) showing similar efficiency as the rabbit reductase was also used to reconstitute solubilized P450s from both plant and mammalian sources.

Reconstitution of P450 activities was performed by the procedure of Laethem and co-workers (25). Typical incubations contain 50 pmol of P450, 50 pmol of NADPH cytochrome P450 reductase, 200 pmol of cytochrome b5, 15 µg of dilauroylphosphatidylglycerol, and 100 µM substrate. Reactions were started by adding NADPH (1 mM final concentration) with the regenerating system noted above. The specific activity of the CYP4As with lauric acid was 8 nmol/min/nmol of P450. Metabolites generated were identified as omega - and (omega -1)-hydroxylauric acids in a molar ratio of 17:1, respectively. CYP2B4 generated a mixture of laurate metabolites hydroxylated at the omega , omega -1, and omega -2 positions (1:5:0.7, respectively) with a specific activity of 800 pmol/min/nmol of P450.

Chemical Labeling

Microsomal incubates containing [1-14C]DDYA were extracted twice with 1 ml of cold acetone to remove unreacted substrate and metabolites. Acetone-insoluble protein (200-400 µg) was collected by centrifugation (15 min at 3000 × g), dissolved in 40 µl of Tris buffer (180 mM, pH 6.8) containing 5% (v/v) SDS, 5% (v/v) beta -mercaptoethanol, 30% (v/v) glycerol, and 0.025% (v/v) bromphenol blue, and heated for 1 min in boiling water. Aliquots of this SDS solution (10 µl) were counted by liquid scintillation and analyzed by SDS-PAGE (26) using a 10% acrylamide gel. After Coomassie Blue staining, gels were soaked for 20 min in Amplify and dried. Radioactivity was detected on dry gels after a 1-month exposure of Kodak x-ray films (Hyper Film-beta max, Amersham Corp.) or 8-15 days of exposure using a bioimaging analyzer (Fugix Bas 2000).


RESULTS

Metabolism of 8- and 9-Dodecynoic Acids

Incubations of microsomes from clofibrate-treated V. sativa seedlings with [1-14C]8- and 9-dodecynoic acids generated two metabolites (Fig. 2, A and B). The metabolites generated either from 8-DDYA or 9-DDYA show similar RF values of 0.35 (Fig. 2, A, peak 1, and B, peak 3) and 0.45 (Fig. 2, A, peak 2, and B, peak 4) on TLC and similar retention times of 11 and 12.5 min by RP-HPLC analysis (not shown). Product formation was NADPH dependent and was inhibited by CO and by antibodies against a NADPH cytochrome P450 reductase isolated from Jerusalem artichoke (Table I). After catalytic hydrogenation and conversion to the corresponding MeTMS derivatives, fractions 1 and 3 (Fig. 2, A and B) generated from either 8- or 9-DDYA showed an MS fragmentation pattern identical to that of the authentic 12-hydroxylaurate MeTMS derivative. Based on HPLC retention times and MS fragmentation analysis, metabolites 1 and 3 were identified as 12-hydroxy-8-dodecynoic and 12-hydroxy-9-dodecynoic acids, respectively (Fig. 1). The structures of metabolites 2 and 4 remain to be determined.

Table I.

Inhibition of microsomal metabolism of a series of acetylenic analogues of lauric acid by carbon monoxide and rat monoclonal antibodies raised against purified cytochrome P450 reductase from Jerusalem artichoke tubers

Metabolites generated were measured as described under "Materials and Methods." Results are means of triplicate incubations. Enzyme activities are expressed as percentages of maximal turnover rates (control) of microsomal enzymes.
Conditions
Metabolism
Antireductase antibodies P450-reductase % activity 8-DDYA 9-DDYA 10-DDYA 11-DDYA

% % % %
None (control) 100a 100b 100c 100d 100e
10 µl 74 79 67 90 82
20 µl 33 70 62 78 53
40 µl 14 30 30 45 17
CO/O2/N2 (2:2:8 v/v/v)
Dark 45 42 37 45
Light 69 60 57 60

a-e Maximal turnover rates: a 53 nmol/min/mg protein for NADPH cytochrome P450-reductase activity and b 31, c 38, d 68, and e 213 pmol/min/mg protein for metabolism of 8-, 9-, 10-, and 11-dodecynoic acids, respectively.

Metabolism of 10-Dodecynoic Acids

When [1-14C]10-DDYA was incubated with microsomes only one metabolite was generated. The reaction product showed a RF of 0.4 on TLC (Fig. 2C, peak 5) and a retention time of 18 min by RP-HPLC analysis. After RP-HPLC isolation and chemical hydrogenation, this product showed a retention time in RP-HPLC similar to authentic 12-hydroxylauric acid (32 min). The identity of the hydrogenated metabolite was further confirmed by GC-MS analysis after conversion to a MeTMS derivative. The mass spectrum was identical to that of authentic 12-hydroxylauric acid (MeTMS). From these results metabolite 5 (Fig. 2C) was identified as 12-hydroxy-10-dodecynoic acid (Fig. 1).

As for 8- and 9-DDYA, metabolism of 10-DDYA was inhibited with both carbon monoxide and antireductase antibodies (Table I), suggesting the involvement of P450 in oxidation of 10-DDYA.

As shown in Table II, the Km values for the acetylenic derivatives were similar (25-110 µM) to that obtained for lauric acid. However, marked differences were noted in Vmax. The Vmax increased as the triple bond in the substrate moved away from the methyl end.

Table II.

Kinetic parameters of metabolism of lauric acid and a series of acetylenic analogues by microsomal fractions from clofibrate-induced V. sativa seedlings


Compounds Kma Vmax(app)a Metabolites

µM pmol/min/mg
Lauric acid 55  ± 2 200  ± 10 12-Hydroxylauric acid
8-DDYA 110  ± 10 162  ± 26 12-Hydroxy-8-DDYA
9-DDYA 87  ± 4 124  ± 13 12-Hydroxy-9-DDYA
10-DDYA 50  ± 25 200  ± 30 12-Hydroxy-10-DDYA
11-DDYA 25  ± 2 20  ± 1 1,12-Dodecandioic acid

a  Mean ± S.D. of triplicate measurements.

Metabolism of [114C]11-Dodecynoic Acid

The efficiency of 11-DDYA to irreversibly inhibit the microsomal omega -LAH activity from V. sativa seedlings was previously demonstrated (10). When microsomes were incubated with [1-14C]11-DDYA, a polar metabolite (Fig. 3A, peak b) was generated. RP-HPLC analysis of the material in peak b showed that it contained two labeled metabolites present in a molar ratio of 1:2.7 (Fig. 3B, peaks 10 and 11). The metabolite in peak 11 was methylated than subjected to mass spectral analysis. The fragmentation pattern of the methyl derivative of peak 11 was similar to that of authentic 1,12-dodecandioic acid dimethyl ester. Ions fragments were at m/z 74 (95%), m/z 87 (41%), m/z 112 (31%), m/z 185 (M-73, 30%), m/z 227 (M-31, 28%), and a base peak at m/z 98. As expected, ion fragments derived from the 14C isotope were increased by 2 atomic mass units and detected at m/z 187 and 229. The metabolite in peak 10 was also isolated by RP-HPLC and subjected to GC-MS analysis. Despite the presence of 14C, we were unable to determine the structure of this product. Howewer, acidic hydrolysis of this metabolite led to 1,12-dodecandioic acid (Fig. 1), suggesting that an unknown nucleophile present in the incubation medium reacted with a reactive intermediate. That this metabolite represents the initial product comes from incubations with [1-14C]11-DDYA as a function of time. The concentration of the unknown metabolite followed the formation of dicarboxylic acid (Fig. 4, A and B). The Kmm(app) for 11-DDYA was 25 µM, and the Vmax(app) was 20 pmol/min/mg, 10 times lower than with laurate (200 pmol/min/mg) as a substrate (Table II).


Fig. 3. Chromatographic resolution of metabolites generated by microsomal incubations from V. sativa seedlings with [1-14C]11-dodecynoic acid. A, radiochromatographic TLC analysis. Microsomal fractions were incubated with [1-14C]11-dodecynoic acid and NADPH in the absence (lower trace) or presence (upper trace) of 10 mM beta -mercaptoethanol at 27 °C. Aliquots (100 µl) of the incubated medium containing 50% of acidified acetonitrile were stopped on TLC layers. Radiolabeled compounds were resolved into 1,12-dodecandioic acid (peak b) and residual 11-dodecandioic acid (peak 9). The metabolite in peak a, which increases with increasing concentration of beta -mercaptoethanol, was not fully identified in the current study but led to 1,12-dodecandioic acid by acidic analysis. B, RP-HPLC elution profile of metabolites contained in peak b of TLC analysis. Metabolites were separated by reverse phase HPLC using a mixture of CH3CN/H2O/CH3COOH (25:75:0.2, v/v/v) as a mobile phase at flow rate of 2 ml/min. The radioactivity was monitored directly with a flow detector. Metabolites generated from [1-14C]11-dodecynoic acid were resolved into unidentified metabolite (peak 10) and 1-12-dodecandioic acid (peak 11). The chemical structure of diacid was resolved by GC-MS analysis.
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Fig. 4. Metabolism of [1-14C]11-dodecynoic acid by V. sativa microsomes as a function of time and concentration. Microsomal fractions were incubated with [1-14C]11-dodecynoic acid in the presence of NADPH at 27 °C for various periods (A) and concentrations (B). Formation of 1,12-dodecandioic acid (black-square; Fig. 2B, peak 11) and identifiable metabolite (open circle ; Fig. 2B, peak 10) was quantified by TLC and RP-HPLC analysis following procedures described in Fig. 2. Results are means of triplicate incubations and expressed as pmol/min/mg of protein.
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As previously reported, 11-DDYA inhibited lauric acid hydroxylation by V. sativa microsomes. The decrease in omega -LAH activity was correlated with the formation of 11-DDYA metabolites (Fig. 5A). When [1-14C]lauric acid, an alternate substrate, and [1-14C]11-DDYA were incubated together, 12-hydroxylauric acid and 1,12-dodecandioic acid were generated in a ratio of about 5:1, respectively. Metabolic activity of the microsomal preparation toward both substrates (lauric acid and 11-DDYA) decreased with time, but the ratio of metabolites remained constant, suggesting that both compounds were metabolized by the same or very similar P450 isoforms (results not shown). Increasing concentrations of lauric acid in the presence of 50 µM [1-14C]11-DDYA resulted in a decrease in 1,12-dodecandioic acid formation and in a parallel increase of the omega -LAH activity, indicating that an alternate substrate protects the plant P450 enzyme against inactivation. Finaly, when microsomal incubation containing 100 µM lauric acid and NADPH was supplemented with increasing concentrations of 1,12-dodecandioic acid (2-50 µM final concentrations), no inhibition of the omega -LAH activity was measured (results not shown). These experiments clearly indicate that the diacid generated from 11-DDYA was not involved in the inhibition of omega -LAH activity.


Fig. 5. A, relationship between production of 11-DDYA metabolites and inhibition of omega -LAH activity as a function of time. Results were from incubations of plant microsomes with 50 µM [1-14C]11-DDYA and cofactors for various periods. The remaining omega -LAH activity (triangle ) was expressed as percentages of maximal activities and the formation of metabolites from 11-DDYA (black-square), which are the sum of 1,12-dodecandioic acid and an unidentified metabolite (see Fig. 3B), in pmol/min/mg of protein. Values are means of triplicate incubations. B, relationship between chemical labeling of protein and formation of metabolites from 11-DDYA as a function of time. Microsomal fractions from clofibrate-induced V. sativa were incubated at 27 °C in the presence of NADPH with 100 µM [1-14C]11-dodecynoic acid for various periods (0-90 min). Chemical labeling of microsomal proteins (triangle ) and metabolites from 11-DDYA (black-square) were subsequently measured as detailed under "Materials and Methods." Radioactivity was quantified by liquid scintillation. Results are expressed as pmol/min/mg of protein, and values are means of triplicate incubations.
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The possibility that enzyme inactivation was the result of heme destruction was investigated. The P450-CO spectra was measured before and after incubation with various 11-DDYA concentrations, and no appreciable changes were observed.

To investigate whether the inhibition was due to covalent modification of the protein, fractions were extracted twice with 1 ml of cold acetone to completely remove residual substrate and metabolites and to precipitate proteins. After solubilization, total radioactivity bound was determined, and an aliquot was analyzed by SDS-PAGE. All four isomeric DDYAs were examined, and only the [1-14C]11-DDYA covalently labeled protein. Compared with tissues from untreated plants, covalent binding was greater in microsomes from clofibrate-treated seedlings, required NADPH, and was inhibited by CO and monoclonal antibodies to NADPH cytochrome P450 reductase (results not shown). Covalent binding was correlated with the dependent formation of 1,12-dodecandioic acid (Fig. 5B).

Autoradiography after SDS-PAGE separation of the inactivated microsomal proteins showed that 70% of the labeling was associated with low molecular weight (LMW) components at the front of the gel. The remaining 30% was associated with proteins with subunit molecular weights of about 53,000 (Fig. 6, lane 2).


Fig. 6. Autoradiography of SDS-PAGE analysis of microsomal proteins from clofibrate-induced V. sativa incubated with [1-14C]11-DDYA: effects of beta -mercaptoethanol and antireductase antibodies on the chemical labeling. Microsomes were incubated with 100 µM [1-14C]11-dodecynoic acid for 30 min with or without NADPH and in the presence of 10 mM beta -mercaptoethanol or monoclonal antibodies raised against purified NADPH cytochrome P450-reductase from H. tuberosus. Metabolites and residual 11-DDYA were extracted prior to SDS-PAGE analysis of sedimented proteins, as described under "Materials and Methods." Gel electrophoresis was stained with Coomassie Blue (not shown) and analyzed by autoradiography. Lanes 1, 3, 5, and 7, incubations without NADPH (control); lanes 2, 4, 6, and 8, incubations with NADPH: NADPH alone (lane 2) and NADPH plus 10 mM beta -mercaptoethanol (lane 4) or NADPH plus 50 and 10 µl of antireductase antibodies (lanes 6 and 8, respectively). The positions of molecular mass markers (kDa) are indicated. Experimental conditions are detailed under "Materials and Methods."
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Effects of Thiol Compounds

The most plausible mechanism for generation of 1,12-dodecandioic acid, the final reaction product, from 11-DDYA is via a reactive ketene intermediate. Alkylation by the electrophile could occur in or out of the enzyme active site. We therefore incubated microsomes with 11-DDYA in the presence of nucleophilic compounds such as nucleophile amino acids, GSH, and beta -mercaptoethanol. The two latter compounds strongly reduced diacid formation. Cysteine was the only amino acid to produce similar inhibition. RP-HPLC or TLC analysis of metabolites generated by incubates containing beta -mercaptoethanol showed the formation of an additional metabolite (Fig. 3A, peak a). The formation of this metabolite increased with increasing concentrations of beta -mercaptoethanol and paralleled the decrease in diacid formation (results not shown). The metabolite was isolated by RP-HPLC, and after acid hydrolysis (4 N HCl, 12 h) 1,12-dodecandioic acid was released. Similar results were obtained using both cysteine and glutathione. SDS-PAGE analysis also showed that the labeled protein was greatly reduced (Fig. 6, line 4) when compared to incubation without beta -mercaptoethanol (Fig. 6, lane 2). At least two protein bands remained labeled, but the nonspecific labeling disappeared. This suggests that the reactive intermediate that mainly diffuses outside of the enzyme site interacts more efficiently with exogenous nucleophile compounds than with protein or water.

Inactivation of Reconstituted Rabbit Liver P450 Isoforms

The mixture of CYP4A isoforms generated mainly 12-hydroxylauric acid and to a lesser extent 11-hydroxylauric acid. The ratio of omega /omega -1 oxidation was 17:1, respectively. CYP2B4 produced a mixture of 12-, 11-, and 10-hydroxylated lauric acids in a molar ratio of 1:5:0.7, respectively.

Incubation of CYP4As with 11-DDYA resulted in a NADPH- and time-dependent loss of lauric acid hydroxylation that occurred with a half-life of 12.8 min. Under the same incubation conditions, 100 µM 10-DDYA did not inactive the CYP4A preparation, although this acetylenic analogue was efficiently converted to 12-hydroxy-10-DDYA (results not shown). No significant loss of spectrally detected P450 was observed. Autoradiography of CYP4A after SDS-PAGE demonstrated that only incubation with the terminal acetylene 11-DDYA resulted in the covalent labeling of proteins when NADPH was present (Fig. 7A, lane 4). A great deal of the radioactivity was detected associated with low molecular weight proteins present in the partially purified preparation.


Fig. 7. Chemical labeling with 10-DDYA and 11-DDYA of P450 forms from rabbit liver in reconstituted activities. Autoradiography of SDS-PAGE gels of a mixture of CYP4A (A) and CYP2B4 (B) incubated for 30 min with [1-14C]10-dodecynoid acid (lanes 1, 2, 5, and 6) and [1-14C]11-dodecynoic acid (lanes 3, 4, 7, and 8) in the absence (lanes 1, 3, 5, and 7) and presence (lanes 2, 4, 6, and 8) of NADPH. The positions of molecular mass markers (Mr × 10-3) are indicated.
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Both 10-DDYA and 11-DDYA inhibited lauric acid hydroxylation by reconstituted CYP2B4 in a NADPH- and time-dependent manner. Enzyme activity was inhibited by 50% with 10-DDYA and 11-DDYA in 15 and 9.8 min, respectively. Autoradiography of inactivated CYP2B4 after SDS-PAGE demonstrated that both mechanism-based inhibitors bind covalently to the hemoprotein (Fig. 7B, lanes 6 and 8). These results strongly contrast with those obtained with P450s from the CYP4A family and show that CYP2B4 produces a metabolite of 10-DDYA that is able to covalently bind to the hemoprotein.


DISCUSSION

Although mammalian fatty acid omega -hydroxylases have been extensively studied, and several of the corresponding genes have been cloned, the catalytic mechanism of these oxygenases is not well established (27, 28, 29). Less is known about the catalytic mechanism and substrate specificity of omega -hydroxylases from plants, enzymes that have not been successfully purified. Among the increasing number of plant P450 cDNAs sequenced to date, none shows greater than 40% similarity to P450s from families 2, 4, 52, and 102, which catalyze fatty acid oxidation in animals, fungi, and bacteria, respectively. This is intriguing from a phylogenetic point of view, since not only substrate specificity but also selective induction by peroxisome proliferators appear similar between plant and animal omega -hydroxylases. One aim of the present work was to probe a better understanding of the mechanism of suicidal inactivation by acetylenes and to determine the relation between the regioselectivity of different fatty acid hydroxylase isoforms and the potency of the external versus internal position of the triple bond in the inhibitor molecule.

The mechanism of inactivation by terminal acetylenes has been extensively studied. It has been reported that the occurrence of heme alkylation versus apoprotein binding is related to oxidative attack at the internal versus terminal carbon of a terminal triple bond (15, 30).

Substrate analogues containing an internal acetylene were omega -hydroxylated by omega -LAH from V. sativa without any measurable enzyme inactivation, corroborating previous results with internal ethylenic laurate analogues (19). These studies had shown that only the methyl end of substrates is accessible to oxidation by V. sativa omega -LAH, and that internal double or triple bonds did not affect the regioselectivity but only kinetic parameters of the hydroxylase. In the present study, two unknown polar metabolites were also generated from 8- and 9-DDYA in addition to the 12-hydroxy derivatives. Inhibition experiments with both CO and antireductase antibodies suggest that these products may be generated by omega -LAH.

In contrast, oxidation of the terminal acetylene (11-DDYA) resulted mainly in the formation of 1,12-dodecandioic acid, which could be formed by the addition of water to a putative ketene intermediate. An unknown minor metabolite was also generated, probably by the same reaction of an endogenous nucleophile other than water with the ketene, since acidic hydrolysis of the metabolite produced the diacid. The time- and concentration-dependent chemical labeling of proteins by 11-DDYA was correlated with the formation of the metabolites, suggesting a partition of the reactive intermediate between diacid formation, alternate product formation, and protein binding causing inactivation of the enzyme (Fig. 8).


Fig. 8. Proposed mechanism of inactivation of omega -LAH from V. sativa by 11- dodecynoic acid. Interactions of a postulated ketene intermediate with heme, apoprotein, and water in the active site of P450 and external interaction of the intermediate with nucleophilic compounds, added or present in the preparation, are shown. A, heme; B, amino acid residue of P450 apoprotein; C, molecule of water generated in the active site; D, external nucleophilic compounds.
[View Larger Version of this Image (29K GIF file)]


In the presence of beta -mercaptoethanol, cysteine, or glutathione the nonspecific labeling of microsomal proteins and diacid formation were strongly decreased, whereas the kinetics of omega -LAH inhibition were not altered. In addition, there was persistent chemical labeling of the Mr 53,000 protein in the presence of thiol reagents. These findings are consistent with a reactive intermediate that inactivates by reaction within the active site, whereas nonspecific reactions with water or proximal protein residues occur after diffusing outside the active site (Fig. 8). The fact that the formation of 1,12-dodecandioic acid was not completely suppressed by nucleophilic reagents suggests that this intermediate might also interact in the active site with a molecule of water generated during catalysis (Fig. 8). In addition, the lack of protection against inactivation of omega -LAH and the persistent chemical labeling of the Mr 53,000 protein despite the presence of thiol reagents suggest that these nucleophiles cannot access the active site. Similar effects of external nucleophiles have been recently reported by Lopez-Garcia and co-workers (31) for inactivation of CYP2C9 by thienilic acid. In contrast, Shirane and co-workers (32) reported that inactivation of P450-BM3 (CYP102) by mechanism-based inhibitors (monothioesters of fatty acids) was completely prevented by glutathione, indicating that the enzyme is inactivated by metabolites that diffuse out of the active site.

The partition ratio between covalent binding of proteins and metabolite formation decreased from about 10 in the absence to 2.5 in the presence of beta -mercaptoethanol. Under these conditions, the distribution of the labeling was 30% associated with Mr 52,000-53,000 proteins and 70% with LMW compounds. This is close to the ratio of about 2 that was found for inactivation of CYP4A1 by 11-DDYA (15). If the LMW compounds comprise heme adducts, a point that remains to be clarified, this would indicate that omega  oxidation of acetylenes may involve heme alkylation, in contrast with the proposal by Ortiz de Montellano and co-workers (15) that addition of activated oxygen to the terminal carbon of acetylene involves protein rather than heme modification.

The effects of internal and terminal acetylenic analogues on V. sativa omega -LAH with the effects on reconstituted activities of purified mammalian forms that oxidize laurate at different positions were compared. Our results show that the internal acetylenes exert an unexpected and highly destructive effect on CYP2B4. In contrast, with the CYP4s, only the terminal acetylene produced covalent binding and irreversible inhibition, in agreement with the results obtained with the V. sativa enzyme. Using a wheat microsome system, we have recently demonstrated that cis-9-octadecen-16-ynoic acid (33) and 10-DDYA (34) irreversibly inhibit the oleic acid and lauric acid (omega -1)-hydroxylases, respectively. In addition, the in vivo inhibition of lauric acid (omega -1)-hydroxylase from wheat by undec-9-yne-1-sulfonic acid confirms the efficacy of suicide substrate inhibitors containing an internal triple bond between the two carbons preferentially attacked by the P450 enzyme (34).

A plausible mechanism, based on the formation of a putative unstable acetylene epoxide would involve: (i) the migration of an alkyl chain resulting from the rearrangement of the acetylene epoxide; and (ii) the formation of a diacid (10-methyl-undecane-1,11-dioic acid) resulting from water addition to an exoketene. In fact, this seems not to be the case, since we could not measure the formation of any diacid from 8-, 9-, and 10-DDYA either with 2B4 or with plant in-chain hydroxylases or P450-BM3 from Bacillus megaterium.

Our results on inactivation of omega -LAH from V. sativa and CYP4A by the terminal acetylene corroborate the mechanism postulated by Ortiz de Montellano and co-workers (15), except for the high degree of labeling of low molecular weight compounds observed with both plant and mammal systems (Figs. 6 and 7). However, following this postulate, inactivation of CYP2B4 by 11-DDYA would mainly result from porphyrin N-alkylation rather than binding to protein. Our results demonstrate that incubation of both internal (10-DDYA) and terminal acetylene (11-DDYA) results in a potent and irreversible inhibition of CYP2B4 and labeling of the enzyme protein. Neither diacid formation nor labeled LMW compounds were observed. The absence of diacid formation contrasts with the results of Roberts and co-workers (35), who demonstrated the formation of 2-naphthylacetic acid and the labeling of CYP2B4 when incubated with 2-ethynylnaphthalene. Moreover, Miller and White (36) recently demonstrated that water is the principal active site nucleophile of CYP2B4. In the present study, the absence of diacid formation suggests that the sequence of events leading to enzyme inactivation could be different from those already described. One alternative is for a direct interaction of an oxirene intermediate with heme or protein. A second possibility is for a very fast and exclusive interaction of a putative ketene with a residue from the active site of CYP2B4. In both cases, either the reactive intermediate is short lived, or the presence of reactive nucleophiles nearby does not allow interaction with water. Finally, covalent binding to heme cannot be excluded, even though no decrease of the spectrometrically P450-CO complex was observed in inactivated CYP2B4 preparations. Our results resemble most those already described for the inactivation of P450SCC by acetylenic steroids. Nagahisa and co-workers (37) did not observed the formation of electrophilic intermediates under conditions that completely inactivated the enzyme.

Finally, in view of the natural presence of acetylenic compounds in several plant species (38, 39, 40), it was of great interest to demonstrate that compounds containing an internal acetylenic function might be metabolized by both plant and mammalian P450-dependent fatty acid omega -hydroxylases. The results show that inactivation of P450 occurs when the acetylenic function is adequately positioned even with isoforms that perform in-chain oxidation.

A second goal of the present work was to devise a means to selectively tag fatty acid hydroxylases as a first step for further isolation and cloning. Very recently, a protein labeled with 11-DDYA was isolated from V. sativa microsomes by successive SDS-PAGE containing increasing concentration of polyacrylamide. The alkylated protein was submitted to in-gel proteolysis, and the resulting peptides were sequenced. By several criteria such as NH2-terminal and heme binding amino acid sequences, we concluded that the alkylated protein corresponded to a cytochrome P450 isozyme. To obtain insight into the substrate specificity, isolation of the corresponding cDNA and its expression in yeast are currently under way.


FOOTNOTES

*   This study has been conducted under the Bioavenir program, Groupe de Recherches "Barrières Cuticulaires," financed by Rhône-Poulenc with the contribution of the Ministère de la Recherche et de l'Espace and the Ministère de l'Industrie et du Commerce Extérieur. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by a fellowship from the Ministère de la Recherche et de la Technologie. To whom correspondence should be addressed: Medical Center North, S-3223, Vanderbilt University Medical School, Nashville, TN 37232. Tel.: 615-343-4316; Fax: 615-343-7156. E-mail: helvigC @ctrvax.vanderbilt.edu.
par    Supported by a BDI fellowship from Rhône-Poulenc.
1    The abbreviations used are: omega -LAH, omega -hydroxylase of lauric acid; CYP, cytochrome P; DDYA, dodecynoic acid; diacid, 1,12-dodecandioic acid; GC-MS, gas chromatography-mass spectrometry; LMW, low molecular weight; MeTMS, trimethylsylil ether methyl ester; RP-HPLC, reverse phase high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
2    N. Tijet and I. Benveniste, unpublished results.
3    I. Benveniste, unpublished results.

Acknowledgments

We are grateful to Prof. J. Capdevila for critically reading the manuscript. We thank Dr. C. Larroque (INSERM U-128) for the generous gift of purified cytochrome b5 from rabbit and Dr. A. Lesot for preparing monoclonal antibodies against NADPH cytochrome P450 reductase from H. tuberosus. We also thank M. F. Castaldi for excellent technical assistance and L. Thiriet for typing the manuscript.


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