substrate binding were analyzed with the program Microsoft Excel.©

Enzyme Activity Studies

The rates of P450BM-3-dependent oxygen and NADPH utilization were measured using 20-50 nM solutions of the enzyme in 50 mM MOPS buffer, pH 7.4, containing 100 µM fatty acid. After a 5-min preincubation, reactions were started by addition of NADPH. Oxygen concentration was measured with a Clark-type oxygen electrode instrument (Yellow Springs Instrument Co.). NADPH concentration was measured spectrophotometrically at 340 nm (epsilon  = 6.22 mM-1 cm-1).

For product quantification and structural characterization, incubations were performed at 30 °C under atmospheric air and with vigorous mixing. Reaction mixtures in 50 mM Tris-Cl buffer, pH 7.4, containing 10 mM MgCl2, 150 mM KCl, 8 mM sodium isocitrate, isocitrate dehydrogenase (1.0 IU/ml), dilauroyl phosphatidyl choline (0.05 µg/ml), and P450BM-3 (2-10 nM, final concentration) were incubated 2.5 min prior to the addition of the sodium salts of either AA, EPA, or ETA (25 mM each in 0.05 mM Tris-Cl buffer, pH 8.0) to final concentrations of 50-100 µM each. After 1 min, reactions were started by the addition of NADPH (1 mM, final concentration). At different time points, aliquots were withdrawn, and the organic soluble products were extracted three times with equal volumes of ethyl ether containing HOAc (0.05%, v/v). After solvent evaporation under a stream of nitrogen, the products were resolved by RP-HPLC on a 5-µm Dynamax Microsorb C18 column (4.6 × 250 mm, Rainin Instruments Co., Woburn, MA) using a linear solvent gradient from 49.9% CH3CN, 49.9% H2O, 0.1% HOAc to 99.9% CH3CN, 0.1% HOAc over 40 min at 1 ml/min. Products were quantified by on-line liquid scintillation using a Radiomatic Flo-One beta -Detector (Radiomatic Instruments, Tampa, FL).

Structural Characterizations

The identification of 18-OH-AA and of 14,15-EET was done using published methodology (23, 24, 25, 26) and confirmed with synthetic standards. Synthetic 16-, 17-, and 18-OH-AAs were resolved by normal phase HPLC on a 5-µm Dynamax Microsorb Silica column (4.6 × 250 mm, Rainin Instruments) using an isocratic solvent mixture composed of 0.4% 2-propanol, 0.1% HOAc, 99.5% hexane at 2 ml/min (Rt ~ 31.5, 33.1, and 36.5 min for 17-, 18-, and 16-OH-AA, respectively). Synthetic 19- and 20-OH-AAs were resolved by NP-HPLC as above using a solvent mixture of 1% 2-propanol, 0.1% HOAc, 98.9% hexane at 3 ml/min (Rt ~ 14.6 and 20.8 min for 19- and 20-OH-AA, respectively). The enantiomers of methyl 14,15-epoxyeicosatrienoate were resolved by chiral phase HPLC as described previously (24).

For the characterization of EPA metabolites, the organic soluble material extracted from solutions containing [1-14C]EPA (100 µM final concentration, 0.1 µCi/µmol), 5 nM P450BM-3, and 1 mM NADPH was resolved by RP-HPLC as described. The radioactive fraction eluting from the HPLC column with the retention time of authentic 17,18-epoxy-EPA (18.7 min) was collected batchwise and further characterized. To confirm the epoxide nature of the metabolite, an aliquot of the purified material (2-5 µg) was incubated, under an argon atmosphere and with constant mixing, with 0.25 ml of a mixture containing 20% EtOH, 40% H2O, and 40% glacial HOAc. After 12 h at room temperature, the reaction mixture was diluted with 1 ml of 0.1 M KCl and extracted twice with equal volumes of ethyl ether. The resulting product co-eluted in RP-HPLC with synthetic vic-17,18-dihydroxy-5, 8,11,14-eicosatetraenoic acid (Rt approx  10 min) and, after derivatization to the corresponding TMS ether, PFB ester, showed a NICI/GC/MS fragmentation pattern identical to that of an authentic standard (Fig. 1A). For regiochemical analysis, an aliquot of the hydrated epoxide (5 µg) was hydrogenated over PtO2, derivatized to the corresponding PFB ester (24), and purified by SiO2 chromatography. The dry residue was dissolved in 200 µl of NaIO4 (10 mg/ml in 70% CH3OH) and, after 2 h at 50 °C, the product was extracted into hexane and purified by RP-HPLC using a linear solvent gradient from 49.9% CH3CN, 49.9% H2O, 0.1% HOAc, to 99.9% CH3CN, 0.1% HOAc over 40 min at 1 ml/min (Rt ~ 43 min). The purified aldehyde, resulting from oxidative cleavage of the vic-diol precursor, was dried under a stream of N2, mixed with 200 µl of 0.5% solution of methoxylamine hydrochloride in pyridine (Pierce), incubated 3 h at 30 °C, extracted into hexane, and then characterized by NICI/GC/MS (Fig. 1B).


Fig. 1. Mass spectral fragmentation properties of the hydration and oxidative cleavage products obtained from the P450BM-3 EPA derived metabolite. HPLC purified samples of the EPA metabolite were hydrated under acid conditions, converted to the corresponding PFB ester TMS ether derivative and analyzed by NICI/GC/MS as described under ``Experimental Procedures'' (panel A). Aliquots of the above hydration product were hydrogenated over PtO2, esterified using pentafluorobenzyl bromine, the vic-diol was cleaved with NaIO4 and, after reaction with methoxylamine hydrochloride in pyridine, the resulting oxime was analyzed by NICI/GC/MS as described under ``Experimental Procedures'' (panel B). Shown are the fragmentation patterns obtained by injecting into the GC column 15 ng of the hydration (Rt ~ 4.98 min, panel A) or 20 ng of the oxidative cleavage product (Rt ~ 10.15 min, panel B). Abscissa, abundance as percent of the base peak; ordinate, m/z.
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For structural analysis, the organic soluble products extracted from solutions containing [1-14C]ETA (100 µM, final concentration, 0.2 µCi/µmol), 2 nM P450BM-3, and 1 mM NADPH were purified by RP-HPLC as above. The radioactive material eluting from the RP-HPLC column between 18 and 20 min was collected batchwise and, after solvent evaporation, resolved into fractions a, b, and c (Rt ~ 16.9, 23.1, and 37.1 min, respectively for a, b, and c) by NP-HPLC on a 5-µm Dynamax Microsorb Silica column (4.6 × 250 mm) using an isocratic solvent mixture composed of 0.5% 2-propanol, 0.1% HOAc, 99.4% hexane at a flow rate of 2 ml/min. After catalytic hydrogenation and derivatization to the corresponding PFB esters, fractions a, b, and c were characterized by NP-HPLC on the above silica column using an isocratic solvent mixture composed of 0.2% 2-propanol, 99.8% hexane at a flow rate of 2 ml/min. In this chromatographic system, the PFB esters of hydrogenated a, b, and c (Rt ~ 20, 23, and 35 min, respectively) co-eluted with the PFB esters of synthetic 17-, 18-, and 19-hydroxyeicosanoic acid, respectively. The PFB esters of hydrogenated a, b, and c were further characterized by NICI/GC/MS.

Stereochemical Analysis of the 18-Hydroxyeicosatetraenoic Acid

To a room temperature mixture of synthetic methyl 18-hydroxyeicosatetraenoate (1.5 mg) (27), R-(+)-alpha -methoxy-alpha -trifluoromethylphenylacetic acid (1.5 mg), and dimethylaminopyridine (0.2 mg) in anhydrous CH2Cl2 (1 ml) was added 1,3-dicyclohexylcarbodiimide (1.5 mg) in one portion with stirring. After 12 h, the solvent was removed in vacuo, and the residue was purified by PTLC (SiO2: 20% EtOAc/hexane, RF approx  0.59). A sample of enzymatically derived 18-hydroxyeicosatetraenoic acid (0.8 mg) was esterified with excess diazomethane in Et2O for 1 h prior to derivatization exactly as described above. The individual Mosher esters were further purified by HPLC using a Dynamax Microsorb Silica column (4.6 × 250 mm) isocratically eluted with 0.35% EtOH, 99.65% hexane at 4 ml/min with UV monitoring at 210 nm. Comparative analysis by co-injection on a Chiracel OD HPLC column (4.6 × 250 mm, J. T. Baker Inc.) isocratically eluted with 0.2% 2-propanol, 99.8% hexane at 2 ml/min with UV monitoring showed the 18(S)-isomer eluted with a retention time of 6.6 min, whereas the 18(R)-isomer and the enzymatically derived product co-eluted with a retention time of 5.8 min.

Stereochemical Analysis of 17,18-Epoxyeicosatetraenoic Acid

A stream of O3 in oxygen was passed for 2 h through a solution of enzymatically generated 17,18-epoxyeicosatetraenoic acid (1.87 mg) in 90% CH3OH, 10% CH2Cl2 cooled to 0 °C. NaBH4 (2 mg) was added, and the mixture was stirred at room temperature. After 30 min, the reaction mixture was diluted with Et2O (2 ml) and H2O (2 ml). The organic phase was separated, and the aqueous phase was extracted once more with Et2O. The combined organic phases were evaporated in vacuo, and the residue was dried azeotropically with anhydrous benzene, then dissolved in dry pyridine (100 µl) to which was added benzoyl chloride (15 µl). After 12 h, the reaction mixture was diluted with CH2Cl2 (2 ml), and a saturated aqueous solution of CuSO4 (300 µl) was added to effect phase separation. The organic layer was collected and concentrated in vacuo, and the residue was purified by PTLC (15% EtOAc, 85% hexane, RF ~ 0.26) using standards of synthetic epoxy-benzoate in adjacent lanes as guides to the location of the enzymatically derived 3,4-epoxyhexan-1-yl benzoate.

A chiral standard of the 3,4-epoxyhexan-1-yl benzoate was prepared by Sharpless asymmetric epoxidation of (3Z)-hexen-1-ol as described previously (28) affording (3R,4S)-epoxyhexan-1-ol, [alpha ]22D +7.27° (c 1.85, CHCl3), as a colorless oil in 45% yield. The epoxy-alcohol (58 mg, 0.5 mmol) was dissolved in dry pyridine (1 ml) and cooled to 0 °C, and benzoyl chloride (58 µl, 0.75 mmol, 1.5 equivalent) was added. After stirring at room temperature for 12 h, the reaction mixture was diluted with CH2Cl2 (10 ml), washed with saturated aqueous CuSO4 solution (3 × 5 ml), brine (5 ml), dried over Na2SO4, and evaporated in vacuo. The residue was purified by PTLC (SiO2: 15% EtOAc, 85% hexane, RF approx  0.26) to give the epoxy-benzoate (98 mg, 100%) as a colorless oil. 1H NMR (CDCl3, 250 MHz): delta  1.06 (t, 3H, 6.6 Hz), 1.49-1.69 (m, 2H), 1.90-2.14 (m, 2H), 2.90-2.98 (m, 1H), 3.09-3.18 (m, 1H), 4.53 (t, 2H, 6.6 Hz), 7.46 (t, 2H, 7.7 Hz), 7.56 (apparent t, 1H, 7.7 Hz), 8.07 (d, 2H, 7.7 Hz). The (3S,4R)-enantiomer was obtained analogously. Comparisons using a Chiracel OC HPLC column (4.6 × 250 mm) eluted isocratically with 0.2% 2-PrOH, 0.1% EtOH, 99.7% hexane at 1.1 ml/min with UV monitoring at 210 nm showed the (3R,4S)-isomer had a retention time of 58 min, whereas the (3S,4R)-enantiomer and the biologically derived sample co-eluted with a retention time of 45 min.

Gas Chromatography-Mass Spectral Analysis

Samples were dissolved in dodecane and analyzed by NICI/GC/MS on a Nermag R1010C quadrupole instrument interfaced to a Varian Vista Gas chromatograph utilizing He and CH4 as carrier and reagent gases, respectively. Splitless injections were made onto a 30-m SPB-1 fused silica capillary column (0.32-mm inner diameter, 0.25-µm coating thickness, Supelco Inc. Bellefonte, PA). After 1 min at 100 °C, the oven temperature was raised to 300 °C at 10 °C/min.


RESULTS AND DISCUSSION

Rates of AA Oxidation by P450BM-3

The rate of fatty acid oxidation by P450BM-3 can be easily measured either polarographically using an oxygen electrode or spectrophotometrically monitoring absorbance changes at 340 nm. Seen in Fig. 2 is an oxygen electrode trace of O2 consumption during the NADPH-dependent metabolism of AA by P450BM-3. The order of addition of reactants is crucial, i.e. NADPH must be added last to avoid inactivating the reductase domain of P450BM-3 (3, 29). During the first few seconds after addition of NADP, the turnover number of P450BM-3 is 3.5 µmol of O2 consumed/min/nmol, but decreases as oxidized products accumulate (Fig. 2). In experiments not shown here, the rate of NADPH oxidation, measured at 340 nm, was approximately the same as the rate of oxygen consumption per mol of P450 per min indicating a tight coupling of NADPH oxidation, O2 consumption and substrate oxidation.


Fig. 2. Oxygen consumption catalyzed by P450BM-3 during the metabolism of AA. P450BM-3 (0.04 µM) was added to the oxygen electrode vessel at 25 °C containing MOPS buffer (50 mM, pH 7.4). The solution was preincubated for approximately 1 min prior to the addition of AA (100 µM). The reaction was initiated with NADPH (1 mM).
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Within the first 30 s of incubation, the rates of AA utilization and product formation began to decrease showing the lack of a clear linear relationship between product formation and incubation time. As the rate of AA oxidation decreased, the recovery of polyoxygenated products, derived from secondary oxygenations, increased concomitantly, and became predominant 2-3 min after initiation. When limiting amounts of AA were added, as shown in Fig. 2, the ratio of O2 consumed per mol of AA added was approximately 2, indicating that fatty acid polyoxygenation had occurred as shown for palmitic acid (5, 7). The limited solubility of AA made impractical attempts to increase its concentration, and thus prolong enzyme-substrate saturation. The rates of product formation shown in Table I were obtained at 30 °C and after a 30 s incubation, and are approximations of the initial velocities. They are useful only for comparative purposes.

Table I.

Reaction rates and spectral binding constants of P450BM-3 using various substrates


Substrate Reaction ratea Spectral binding constant, Ks

µM
AA 3.2  ± 0.4 1.2  ± 0.1
EPA 1.4  ± 0.2 1.6  ± 0.5
ETA 2.9  ± 0.1 NDb

a  Reaction rates in µmols of product formed/min/nmol of P450BM-3.
b  Not determined.

Among the fatty acids metabolized by P450BM-3, AA showed the highest oxidation rate, 3.2 µmol of product formed/min/nmol of P450BM-3 (Table I). To the best of our knowledge, the rates at which P450BM-3 catalyzes the redox coupled activation of molecular oxygen, the cleavage of the oxygen-oxygen bond, and the insertion of a reactive oxygen atom into the AA molecule are the highest ever reported for a mixed function oxidase, and in particular for a P450, NADPH-dependent, catalyzed reaction. It is of interest that these rates of metabolism (kcat approx  50 s-1) are similar to those of the electron transfer from NADPH to the FMN cofactor of the reductase domain of P450BM-3 (k approx  80 s-1) (29). More importantly, as shown below, under the reaction conditions employed, P450BM-3 displayed high regiochemical selectivity for the fatty acid omega -2 carbon and 14,15-olefinic bond, and generated 18-OH-AA and 14,15-epoxyeicosatrienoic acid (14,15-EET) as major reaction products (80 and 20% of the total products, respectively) (Table II). While small and variable amounts of 17-OH-AA were also formed, they accounted for less than 1% of the total products.

Table II.

Oxidation of AA by P450BM-3 and chiral characterization of the products

P450BM-3 was incubated with [1-14C]AA (1-5 µCi/µmol) NADPH (5 nM, 100 µM, and 1 mM, final concentration, respectively) and an enzymatic NADPH regenerating system. After 30 s at 30 °C, organic soluble products were extracted into acidified ethyl ether, resolved by RP-HPLC, and quantified by on-line liquid scintillation as described under ``Experimental Procedures.'' Values are averages ± standard error of the mean, calculated from at least four different experiments.
Product Reaction ratea Product distribution Chirality Chiral distribution

% %
18-OH-AA 2.6  ± 0.2 80 18(R)-OH-AA 96
18(S)-OH-AA 4
14,15-EET 0.6  ± 0.1 20 14(R),15(S)-EET 1
14(S),15(R)-EET 99

a  Reaction rates in µmol of product formed/min/nmol of P450BM-3. The total rate of metabolite formation was 3.2 ± 0.4 µmol/min/nmol of P450BM-3.

At difference with AA, the rates of epoxygenation of EPA at 17,18-olefinic bond remained constant within the first 2 min of incubation. Thus, initial velocities were measured 1 min after NADPH addition. However, as with AA, within the first 30 s of incubation at 30 °C, the relationship between the incubation time and the extent of ETA oxidation was nonlinear. Consequently, the reaction rates shown in Table I, were obtained after 30 s and are given as estimates of the initial rates and are useful only for comparative purposes. As with AA and palmitic (5, 7) acid, extended incubation times (2-5 min) resulted in polyoxygenation of ETA.

Spectral Binding Constants for P450BM-3

To assess the binding of AA and EPA to P450BM-3, the enzyme was titrated with substrate, and absorbance changes in the Soret region were measured. As shown in Fig. 3, the AA-dependent decrease in absorbance at 418 nm and the increase in absorbance at 390 nm is indicative of substrate binding to P450s and the conversion of the heme iron from the low to the high spin state (30). The Ks for AA binding to P450BM-3 is 1.2 ± 0.1 µM and that for EPA 1.6 ± 0.5 µM (Fig. 3, Table I). Both have approximately the same binding constants as those of saturated fatty acids (3, 5); however, the latter substrates are oxidized at a substantially lower rate than the eicosanoids. (The Ks of ETA was not determined because of its limited availability.)


Fig. 3. Absorbance spectral changes on AA binding to P450BM-3. P450BM-3 (0.5 µM) in MOPS buffer (50 mM, pH 7.4) was titrated with a freshly prepared solution of AA and the absorbance changes recorded following each addition (0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 9, 11, and 16 µM, final concentration). The inset is a plot of 1/[ES] versus 1/[Si] where [ES] was calculated from the change in absorbance at 393 nm using the equation [ESi] = Delta Ai × [P450]/Delta Amax and Delta Amax as the maximum absorbance change at 393 nm; [Si] = [Stotal- [ESi]. Not all spectra used for the calculation of Ks are shown.
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AA Oxidation by P450BM-3

Incubations of AA with P450BM-3 resulted in the rapid, NADPH-dependent formation of two predominant metabolites with RP-HPLC retention times of 15.5 and 22.6 min (Fig. 4, fractions A and B). Additional amounts of a radiolabeled product (<1% of the total products), with a retention time identical to authentic 17-OH-AA (approximately 14.5 min) were recovered. To minimize secondary metabolism due to the unusually high reaction rates, the hemoprotein and the fatty acid concentrations were maintained at <= 5 nM and >= 50 µM, respectively. For the first 2 min of incubation under these conditions, primary oxygenation products were recovered almost exclusively, i.e. derived from the insertion of a single atom of atmospheric oxygen. Higher enzyme concentrations, longer incubation times, and/or lower fatty acid concentrations led to complex product profiles resulting from reiterative oxygenation of primary metabolites. Among these products, 14,15-epoxy-18-hydroxyeicosatrienoic acid was identified by GC/MS.


Fig. 4. Chromatographic resolution of the products generated by P450BM-3 during the metabolism of AA. The organic soluble products generated by solutions containing P450BM-3 (5 nM), [1-14C]AA (100 µM) and NADPH (1 mM), were resolved by RP-HPLC as described under ``Experimental Procedures.'' Shown is the radiochromatogram derived from a 30-s solution that contained a total of 2 pmol of P450BM-3.
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For structural analysis, the reaction products shown in Fig. 4 were collected and, after solvent evaporation, characterized utilizing a combination of functionality-specific chemical derivatization reactions and chromatographic and NICI/GC/MS techniques. The reaction product, with a RP-HPLC elution time of 15.5 min (Fig. 4, fraction A), was conclusively identified as 18-OH-AA based on the following: 1) co-elution on reversed and normal phase HPLC with an authentic standard, 2) co-elution of its PFB-TMS derivative on capillary GC with a similarly derivatized standard, 3) NICI/GC/MS analysis of its PFB-TMS derivative showed the presence of an intense ion fragment at m/z 407 (loss of PFB, base peak) indicative of a monohydroxylated metabolite, 4) catalytic hydrogenation followed by conversion to the corresponding PFB-TMS derivative yielded a product that co-eluted on capillary GC with a similarly derivatized authentic standard, and 5) NICI/GC/MS analysis of its hydrogenated PFB-TMS derivative yielded a fragment ion at m/z 415 (loss of PFB, base peak), consistent with the presence of four double bonds in the parent molecule that had been reduced upon hydrogenation.

Fraction B (Fig. 4, retention time 22.6 min) was shown to contain 14,15-EET based on the following chromatographic and mass spectral evidence: 1) co-elution in reversed and normal phase HPLC with authentic 14,15-EET (23, 24); 2) co-elution in capillary GC of its PFB ester derivative with authentic 14,15-EET-PFB (23, 24); 3) the NICI mass spectrum of the PFB ester of fraction B was identical to that of synthetic 14,15-EET-PFB with major ion fragments at m/z: 319 (base peak, loss of PFB), 303 (20% of base peak, loss of PFB and oxygen) and 301 (12% of base peak, loss of PFB and water) (24); and 4) the NICI mass spectrum of the PFB ester of hydrogenated B was nearly identical to that of the PFB ester of authentic 14,15-epoxyeicosanoic acid with major ion fragments at m/z: 325 (base peak, loss of PFB), 309 (10% of base peak, loss of PFB and oxygen) and 307 (8% of base peak, loss of PFB and water) (24, 26).

Hydroxylation of saturated fatty acids (e.g. palmitic acid) by P450BM-3 occurs preferentially at the omega -2 carbon atom, with omega -1 and omega -3 hydroxylated products accounting for a substantial portion of the total metabolism (3, 5). In contrast, P450BM-3 hydroxylates the AA C18 carbon in a nearly exclusive fashion, i.e. 18-OH-AA accounts for better than 99% of the overall sp3 carbon hydroxylation. Hydroxylation at the fatty acid omega -3 carbon accounted for less than 1% of the total products and no metabolism was observed at the omega -1 or omega  carbons (Fig. 5). Even though amino acid sequence analysis indicates a 25-30% sequence similarity between P450BM-3 and CYP4A gene subfamily isoforms (a group of mammalian microsomal fatty acid omega -hydroxylases) (4), none of the CYP4A isoforms catalyzes fatty acid or AA omega -2 oxidation or epoxidation (11, 13). However, the formation of 18-OH-AA and of 18(R)-OH-AA by rat hepatic and monkey seminal vesicle microsomal fractions, respectively, has been reported (31). Recent studies have also shown that hydroxylation at the C-18 position of AA was catalyzed by CYP1A1, CYP1A2, and CYP2E1 (11, 13, 25). Hydroxylations at the sp3 carbons near or at the fatty acid methyl end require the delivery by the protein catalyst of a reactive, heme-bound oxygen species to a ground state sp3 carbon atom. It is therefore likely that for all these reactions the oxygen chemistries and the mechanism(s) are similar yet independent of the fatty acid chain length and/or degree of saturation. Nevertheless, compared to saturated fatty acids, the AA molecule imposes additional steric requirements on the active site of the enzyme. During AA C18 hydroxylation, the P450 binding/active site must position the acceptor carbon atom not only in optimal proximity to the heme-bound active oxygen, but also with complete segregation of the fatty acid reactive bis-allylic methylene carbons at C-7, C-9, and C-13 and the 5,6-, 8,9-, 11,12-, and 14,15-olefinic bonds.


Fig. 5. The product distribution from the P450BM-3 catalyzed metabolism of AA and ETA.
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To further delineate the structural determinants responsible for AA active site binding and productive spatial orientation, the chiral properties of the metabolites formed by P450BM-3 were characterized. In previous studies it was shown that AA epoxidation and omega -2 carbon hydroxylation by mammalian P450 isoforms proceeded with a degree of enantiofacial selectivity unprecedented for P450 catalyzed oxidations of acyclic, unbiased molecules such as AA (9, 11, 13). As shown in Table II, P450BM-3 hydroxylates the omega -2 carbon atom of AA in a highly asymmetric mode generating 18(R)-OH-AA with 96% optical purity. Similarly, P450BM-3 catalyzed AA epoxidation yields 14(S),15(R)-EET with 99% optical purity. It thus appears that P450BM-3 evolved a highly structured and spatially rigid substrate binding site, capable of accommodating a polyunsaturated fatty acid such as AA in optimal orientation with regards to the heme-bound active oxygen. The unprecedented high enantiofacial selectivity of this protein, in conjunction with its moderate regioselectivity, indicates that during catalytically productive binding: 1) the AA carbon-carbon rotational freedom is substantially restricted, and 2) the active site spatial coordinates allow for a moderate degree of substrate lateral displacement. Finally, while 18(R)-OH-AA is the predominant enantiomer formed by mammalian microsomal enzymes (31), 14(S),15(R)-EET is the predominant enantiomer found in vivo in rat liver, plasma, and kidney (26, 32, 33).

EPA and ETA Oxidation by P450BM-3

The metabolism of EPA and ETA, two AA analogs (Fig. 5), was studied to probe the role that substrate structural features, in particular C-H bond acceptor chemistry, play as determinants of P450BM-3 catalytic outcome. We selected these AA analogs because: 1) radiolabeled EPA and limited amounts of radiolabeled ETA were commercially available, 2) all of the molecules are of the same carbon length, 3) between carbons C-1 and C-13, all three fatty acids (AA, EPA, and ETA) are structurally identical, 4) ETA, with its sp3 C-14-C-15 carbons, was used to probe the role of the AA 14,15-olefinic bond in inducing oxygenation of C-H bonds distal to the fatty acid omega -2 carbon, the substrate's most metabolically active carbon atom (Table II), and 5) EPA, with the extra olefinic bond at C-17-C-18 allowed for the analysis of C-H bond acceptor reactivity in the enzyme's regioselectivity of oxygen insertion.

Incubation of EPA with P450BM-3 and NADPH resulted in the time dependent formation of a radioactive product with a RP-HPLC retention time of 18.7 min (Fig. 6). Importantly, and at difference with AA, longer incubation times (>= 4 min) and/or higher enzyme concentrations (>= 10 nM) did not result in substantial changes in the profile shown in Fig. 6, indicating that the EPA oxygenated metabolite is a poor substrate for P450BM-3. Structural analysis demonstrated that the sole product of EPA metabolism by P450BM-3 was 17,18-epoxyeicosatetraenoic acid (17,18-epoxy-EPA), i.e. no other product was generated during the first 3 min of incubation at 30 °C; however, during prolonged incubations, the 17,18-epoxy-EPA underwent partial chemical hydration to 17,18-dihydroxyeicosatetraenoic acid. Additionally, as shown in Table I, EPA was metabolized at approximately half the rate estimated for AA. On the other hand, the EPA 17,18-olefinic bond is epoxidized at more than double the rate of the AA 14,15-olefinic bond (Tables II and III). Significantly, under the experimental conditions used, epoxidation of the EPA 14,15-olefinic bond was negligible (less that 1% of the total reaction products). The identification of the P450BM-3 metabolite as 17,18-epoxy-EPA was based on the following: 1) co-elution in reversed and normal phase HPLC with an authentic standard; 2) co-elution in capillary GC of its PFB ester with a similarly derivatized standard; 3) the mass spectrum of its PFB ester, under NICI conditions, was nearly identical to that of authentic 17,18-epoxy-EPA with major ion fragments at m/z: 317 (base peak, loss of PFB), 301 (16% of base peak, loss of PFB and O) and 299 (22% of base peak, loss of PFB and H2O); 4) acid catalyzed hydrolysis (in 50% HOAc) yielded a radioactive product with RP-HPLC characteristics identical to that of synthetic 17,18-dihydroxyeicosatetraenoic acid; 5) the NICI/GC/MS spectra of the PFB ester-TMS ether derivative of the metabolite hydration product matched that of a similarly derivatized standard with major ion fragments at m/z: 479 (base peak, loss of PFB), 407 (25% of base peak, loss of PFB and TMSO), 317 (15% of base peak, loss of PFB, TMS, and TMSOH) (Fig. 1A); 6) acid catalyzed hydrolysis followed by catalytic hydrogenation and NaIO4 oxidative cleavage of the diol carbon-carbon bond afforded a radioactive aldehyde with a RP-HPLC retention time identical to that of the synthetic PFB ester of 17-oxo-heptadecanoic acid; and 7) incubation of the above aldehyde with methoxylamine hydrochloride yielded the corresponding PFB ester-methoxime derivative with a diagnostic molecular weight of 313 (Fig. 1B).


Fig. 6. Chromatographic resolution of the products generated by P450BM-3 during the metabolism of EPA. The organic soluble products generated by solutions containing P450BM-3 (2.5 nM), [1-14C]EPA (100 µM) and NADPH (1 mM) were resolved by RP-HPLC as described under ``Experimental Procedures.'' Shown is the radiochromatogram derived from a 2-min solution that contained a total of 1 pmol of P450BM-3. The retention time for fraction A is 18.7 min.
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Table III.

P450BM-3 oxidation of EPA

P450BM-3 was incubated with [1-14C]EPA (1-5 µCi/µmol) NADPH (2.5 nM, 100 µM, and 1 mM, final concentrations, respectively) and an enzymatic NADPH regenerating system. After 30 s at 30 °C, organic soluble products were extracted into acidified ethyl ether, resolved by RP-HPLC, and quantified by on-line liquid scintillation. Absolute configurations were determined by chiral phase HPLC as described in Experimental Procedures.
Product Product distribution Reaction ratea

%
17,18-EPA  >= 99 1.42  ± 0.20
14,15-EPA <1 <0.01
17(R), 8(S)-EPA 3
17(S),18(R)-EPA 97

a  Reaction rates in µmols of product formed/min/nmol of P450BM-3. Values are averages ± standard error of the mean, calculated from at least four different experiments.

For chiral analysis of the 17,18-epoxy-EPA, we initially utilized degradative ozonolysis followed by derivatization to the corresponding 3,4-epoxyhexan-1-yl benzoates. The Chiralcel OC HPLC properties of the synthetic standards were then compared to those of the biologically derived sample (see ``Experimental Procedures'' for further details). For routine nondestructive analysis, the optical antipodes of methyl 17,18-epoxyeicosatetraenoate were resolved with baseline separation by chiral phase HPLC on a Chiracel OB column as shown in Fig. 7. Absolute configurations were assigned based on the results obtained by the above degradative ozonolysis procedure. Chiral analysis of the EPA epoxygenase metabolite demonstrated that its biosynthesis was highly asymmetric and generated 17(S),18(R)-epoxy-EPA with 97% optical purity (Table III). The high degree of regio- and stereochemical selectivity shown by P450BM-3 during the metabolism of EPA, illustrates the key role played by the C-H acceptor in directing catalytic outcome. Thus, assuming similar active site binding coordinates for AA and EPA, the pi  electron cloud associated with the EPA 17,18-olefinic bond is an efficient trap for the heme-bound oxygenating intermediate, more so than that associated with the AA omega -2 sp3 carbon sigma  orbital. Therefore, C-H acceptor chemistry and active site binding coordinates, as opposed to reactive oxygen chemistry and/or heme-oxygen redox properties, provide a coherent explanation for product chemistry. It is of interest that for both the AA 14,15- and EPA 17,18-epoxygenases, oxygen was delivered to the olefinic bond's si,re-face.


Fig. 7. Chiral phase HPLC chromatographic properties of synthetic and enzymatically produced methyl 17,18-epoxyeicosatetraenoate. Racemic samples of synthetic methyl 17,18-epoxy-EPA (4 µg), enzymatically generated methyl 17,18-epoxy-[1-14C]EPA (6 µg, 4.0 µCi/µmol) of a mixture of the synthetic and biological product were resolved using a Chiracel OB column (4.6 × 250 mm) and a solvent mixture of 0.03% 2-propanol, 99.7% hexane at 2 ml/min, with UV detection at 210 nm. For quantification, the enantiomers were collected individually and, after solvent evaporation, their radioactivity determined by liquid scintillation. Absolute configurations were assigned as described in the text.
[View Larger Version of this Image (15K GIF file)]

As mentioned, ETA lacks olefinic bonds in the vicinity of its omega -3 sp3 carbon (i.e. C-17-C-18), as well as at C-14-C-15 (Fig. 5), and as a result the carbons at or near its methyl terminus are chemically similar to those of most saturated fatty acid substrates. Incubations of [1-14C]ETA (50-100 µM) with P450BM-3 resulted in the NADPH-dependent formation of radioactive metabolites with an average HPLC retention time of approximately 19.3 min (Fig. 8, fraction A). The broad, asymmetric nature of fraction A (Fig. 8) suggested the presence of more than one product. Fraction A was collected from the HPLC eluate and resolved by normal phase HPLC into three radioactive metabolites (Fig. 9). After HPLC purification, fractions a, b, and c (Fig. 9) were submitted to catalytic hydrogenation, derivatized to the corresponding PFB esters TMS ethers and their chromatographic and NICI/GC/MS properties were compared to those of the synthetic PFB esters of 16-, 17-, 18-, 19-, and 20-hydroxyeicosanoic acids. Under NICI conditions, the PFB ester, TMS ether derivatives of hydrogenated a, b, and c yielded similar MS/fragmentation patterns, with a common predominant ion fragment at m/z 393 (M-PFB, base peak). These mass spectra were nearly identical to those obtained with the PFB esters, TMS ether derivatives of isomeric synthetic 17-, 18-, 19-, and 20-hydroxyeicosanoic acids. Fractions a, b, and c were identified as 17-, 18-, and 19-hydroxyETAs based on the following: 1) after catalytic hydrogenation, the PFB ester of a, b, and c co-eluted in normal and RP-HPLC with the PFB ester of authentic 17-, 18-, and 19-hydroxyeicosanoic acid, respectively (Rt ~ 20, 23, and 35 min for the PFB esters of 17-, 18-, and 19-hydroxyeicosanoate, respectively); and 2) the GC retention time for the PFB ester, TMS ether derivatives of hydrogenated a, b, and c corresponded to those of similarly derivatized samples of synthetic 17-, 18-, and 19-hydroxyeicosanoic acid, respectively. The limited supply of [1-14C]ETA precluded a detailed chiral analysis of the metabolites generated by the P450BM-3 catalyzed oxidation of ETA.


Fig. 8. Chromatographic resolution of the products generated by P450BM-3 during the metabolism of ETA. The organic soluble products generated by solutions containing P450BM-3 (5 nM), [1-14C]ETA (100 µM) and NADPH (1 mM), were resolved by RP-HPLC exactly as described in Fig. 4. Shown is the radiochromatogram derived from a 1-min solution that contained a total of 1 pmol of P450BM-3. The radioactive materials in fraction A eluted between 18.2 and 19.7 min.
[View Larger Version of this Image (9K GIF file)]


Fig. 9. Chromatographic resolution of the ETA metabolites generated by P450BM-3. The 1-14C-labeled metabolites (0.2 µCi/µmol) eluting from the RP-HPLC column between 18 and 20 min (Fig. 8) were collected batchwise and, after solvent evaporation, resolved by normal phase HPLC on a 5-µm Dynamax Microsorb Silica column utilizing an isocratic solvent mixture composed of 99.4% hexane, 0.5% 2-propanol, and 0.1% HOAc at 2 ml/min. The column eluent was monitored, on-line, for UV absorbance at 210 nm (top) and radioactivity as in Fig. 4 (bottom). The retention times for fractions a, b, and c were 16.9, 23.1, and 37.1 min, respectively.
[View Larger Version of this Image (14K GIF file)]

For ETA, as was the case for AA and palmitic acid, extended incubation times (>= 2 min) resulted in further metabolism of 17-, 18-, and 19-hydroxy-ETA. Among the secondary oxidation products, 18-oxo-ETA was identified using GC/MS. As shown in Table I, P450BM-3 metabolized ETA at rates comparable to those obtained with AA. Under conditions favoring primary metabolism, the enzyme generated 17-, 18-, and 19-hydroxy-ETA in a 2.4:2.2:1 molar ratio, respectively (Table IV). Importantly, the moderate degree of regioselectivity of the enzyme for the ETA omega -1, omega -2 or omega -3 sp3 carbon atoms is similar to that previously obtained with several saturated fatty acid substrates (5, 34).

Table IV.

P450BM-3 oxidation of ETA

P450BM-3 was incubated with [1-14C]ETA (1-5 µCi/µmol) NADPH (5 nM, 100 µM, and 1 mM final concentrations, respectively) and an enzymatic NADPH regenerating system. After 30 s at 30 °C, organic soluble products were extracted into acidified ethyl ether, resolved by RP-HPLC and quantified by on-line liquid scintillation. Product distribution and rates of individual metabolite formation were obtained after normal phase resolution (Fig. 9) of the radioactive material eluting from the RP-HPLC column with a retention time of 18.6 min (Fig. 8). See ``Experimental Procedures'' for further details.
Product Product distribution Reaction ratea

%
Total 100 2.9  ± 0.10
17-OH-ETA 43 1.2  ± 0.04
18-OH-ETA 39 1.1  ± 0.1
19-OH-ETA 18 0.5  ± 0.06

a  Reaction rates in micromoles of product formed/min/nmol of P450BM-3. Values are averages ± standard error of the mean calculated from at least three different experiments.

Thus, for all the polyunsaturated fatty acids tested here, the preferred sites for the P450BM-3 catalyzed oxygen insertion were the omega -2 and omega -3 carbon atoms. P450BM-3 metabolized the omega -2 or the omega -3 carbons of ETA at rates comparable to those of EPA epoxidation (in micromoles of product/min/nmol of P450: 1.2 and 1.1 for 17- and 18-OH-ETA, and 1.4 for 17, 18-epoxy-EPA, respectively) (Tables III and IV). On the other hand, 19-OH-ETA and 14,15-EET are generated at lower rates (Tables II and IV). It is therefore likely that all three molecules occupy more or less similar spatial coordinates in the active site of P450BM-3.

Modeling AA Binding to the Active site of P450BM-P

Only the atomic coordinates of the substrate-free form of P450BM-P are known (19). Attempts to soak either AA or palmitic acid into preformed crystals of P450BM-P to obtain crystals of substrate bound enzyme has resulted in either low occupancy of the active site by the substrate fatty acid or disorder in the crystals.2 Thus, to enable us to visualize substrate binding in the active site of P450BM-3, we have utilized a molecular modeling approach in which AA was ``docked'' into the substrate access channel and active site. The fatty acid carboxylate was positioned within charge coupling distance of the guanidinium group of Arg47 at the mouth of the access channel (19). The remainder of the AA molecule was built into the volume of the substrate access channel defined by Arg47 at the surface and Phe87 above the heme, maintaining the appropriate bond angles and with reduced van der Waals contacts with amino acid side chains and backbone atoms. During energy minimization of AA in the substrate access channel and active site, Phe87 moved slightly allowing the omega -end of the AA molecule to occupy more readily an existing active site cavity (Fig. 10). In this model, the omega -end is bent upward and toward the fatty acid 14,15-olefinic bond. The pro-R hydrogen of C-18 of AA was closest to the heme iron. An additional displacement of F87 would position the AA 14,15-olefinic bond in optimal proximity to the P450BM-3 heme iron. We propose that AA bound P450BM-3 oscillates between these two alternate conformers in response to the positioning of Phe87 with respect to the heme iron. Clearly AA must be as close to the heme iron of P450BM-3 as depicted in Fig. 10 to exhibit the stereochemical oxidation of AA described here.


Fig. 10. Model of AA bound to P450BM-P.
[View Larger Version of this Image (103K GIF file)]

In conclusion, while epoxidation and a combination of epoxidation and sp3 hydroxylation have been demonstrated in mammalian P450s of the CYP1 and CYP2 families (9, 11, 12, 25, 35, 36), with highly enantioselective epoxidations catalyzed by CYP2C11 and CYP2C23 (36, 37), none of the characterized eukaryotic AA epoxygenases appears capable of epoxidizing a single AA olefinic bond with complete exclusion of the other three. The present study has demonstrated that the common reactive oxygen intermediate that has been proposed for P450 monooxygenations (38) can catalyze epoxidation and sp3 carbon hydroxylation of AA to 18-OH-AA and 14,15-EET (80 and 20% of total products, respectively) in the reaction catalyzed by P450BM-3. In addition, by comparing the stereoselective epoxidation of EPA at the 17,18-olefinic bond, and by contrast, the less selective 17-, 18-, and 19-hydroxylation of ETA with that of AA, we find that in contradistinction to the nature and chemical properties of the reactive oxygen intermediate(s), the chemistry of the reaction products are critically dependent on: 1) the chemical properties of the acceptor C-H bonds and 2) the optimal orientation of the C-H acceptor with respect to the heme-bound reactive oxygen intermediate. We also can conclude from these stereoselective oxidations of AA and EPA that the active site geometry responsible for substrate binding and orientation must restrict the freedom of substrate C-C bond rotation while, at the same time, allowing some degree of substrate lateral mobility that, for P450BM-3 permits 14,15-EET formation. This substrate lateral mobility may be controlled by the positional relationship of the active site residue Phe87 to the heme iron. Thus, substrate chemistry and protein structural features, as opposed to oxidant chemistry, are the key determinants of catalytic outcome. Finally, in as much as the biological function(s) of P450BM-3 are yet to be determined, the high catalytic rates and unprecedented degree of regio- and stereochemical selectivity displayed by the enzyme during the metabolism of AA and EPA are indicative of evolutionary specialization and suggest a role for P450BM-3 in the metabolism of bacterial unsaturated fatty acids.


FOOTNOTES

*   This research was supported in part by National Institutes of Health Grants GM43479 (to J. A. P.), GM37922 (to J. H. C.), and GM31278 (to J. R. F.). 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.
''   To whom correspondence should be addressed: Dept. of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9038. Tel.: 214-648-2361; Fax: 214-648-8856; E-mail: peters01{at}UTSW.SWMED.EDU.
1   The abbreviations used are: P450BM-3, CYP102, the soluble, bacterial P450 isolated from Bacillus megaterium; P450BM-P, the hemoprotein domain of P450BM-3; EET, cis-epoxyeicosatrienoic acid; EPA, eicosapentaenoic acid; ETA, eicosatrienoic acid; 16-, 17-, 18-, 19-, and 20-OH-AA, 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acid, respectively; HPLC, high pressure liquid chromatography; RP, reverse phase; NP, normal phase; PTLC, preparative thin layer chromatography; GC, gas chromatography; MS, mass spectrometry; NICI, negative ion electron capture chemical ionization; Me, methyl ester; TMS, trimethylsilyl ether; PFB, pentafluorobenzyl ester; MOPS, 3-(N-morpholino)propanesulfonic acid; Rt, retention time.
2   S. S. Boddupalli, K. G. Kurumbail, J. A. Peterson, and J. Deisenhofer, unpublished observations.

REFERENCES

  1. Miura, Y., Fulco, A. J. (1974) J. Biol. Chem. 249, 1880-1888 [Abstract/Free Full Text]
  2. Miura, Y., Fulco, A. J. (1975) Biochim. Biophys. Acta 388, 305-317 [Medline] [Order article via Infotrieve]
  3. Narhi, L. O., Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169 [Abstract/Free Full Text]
  4. Wen, L.-P., Fulco, A. J. (1987) J. Biol. Chem. 262, 6676-6682 [Abstract/Free Full Text]
  5. Boddupalli, S. S., Estabrook, R. W., Peterson, J. A. (1990) J. Biol. Chem. 265, 4233-4239 [Abstract/Free Full Text]
  6. Peterson, J. A., Mock, D. M. (1975) Cytochromes P450 and b5 (Cooper, D. Y., Rosenthal, O., Snyder, R., Witmer, C., eds) , p. 311, Plenum Press, New York
  7. Boddupalli, S. S., Pramanik, B. C., Slaughter, C. A., Estabrook, R. W., Peterson, J. A. (1992) Arch. Biochem. Biophys. 292, 20-28 [Medline] [Order article via Infotrieve]
  8. Smith, W. L. (1992) Am. J. Physiol. 263, F181-F191 [Abstract/Free Full Text]
  9. Capdevila, J. H., Falck, J. R., Estabrook, R. W. (1992) FASEB J. 6, 731-736 [Abstract/Free Full Text]
  10. McGiff, J. C. (1991) Annu. Rev. Pharmacol. Toxicol. 31, 339-369 [CrossRef][Medline] [Order article via Infotrieve]
  11. Oliw, E. H. (1994) Prog. Lipid Res. 33, 329-354 [CrossRef][Medline] [Order article via Infotrieve]
  12. Harder, D. R., Campbell, W. B., Roman, R. J. (1995) J. Vasc. Res. 32, 79-92 [Medline] [Order article via Infotrieve]
  13. Capdevila, J. H., Zeldin, D., Makita, K., Karara, A., Falck, J. R. (1995) Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., eds) , 2nd Ed. , p. 443, Plenum Press, New York
  14. Imaoka, S., Tanaka, S., Funae, Y. (1989) Biochem. Int. 18, 731-740 [Medline] [Order article via Infotrieve]
  15. Roman, L. J., Palmer, C. N., Clark, J. E., Muerhoff, A. S., Griffin, K. J., Johnson, E. F., Masters, B. S. (1993) Arch. Biochem. Biophys. 307, 57-65 [CrossRef][Medline] [Order article via Infotrieve]
  16. Cupp-Vickery, J. R., Poulos, T. L. (1995) Nat. Struct. Biol. 2, 144-153 [Medline] [Order article via Infotrieve]
  17. Poulos, T. L., Finzel, B. C., Howard, A. J. (1987) J. Mol. Biol. 195, 687-700 [Medline] [Order article via Infotrieve]
  18. Hasemann, C. A., Ravichandran, K. G., Peterson, J. A., Deisenhofer, J. (1994) J. Mol. Biol. 236, 1169-1185 [Medline] [Order article via Infotrieve]
  19. Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., Deisenhofer, J. (1993) Science 261, 731-736 [Medline] [Order article via Infotrieve]
  20. Boddupalli, S. S., Oster, T., Estabrook, R. W., Peterson, J. A. (1992) J. Biol. Chem. 267, 10375-10380 [Abstract/Free Full Text]
  21. Oster, T., Boddupalli, S. S., Peterson, J. A. (1991) J. Biol. Chem. 266, 22718-22725 [Abstract/Free Full Text]
  22. Omura, T., Sato, R. (1964) J. Biol. Chem. 239, 2370-2378 [Free Full Text]
  23. Capdevila, J. H., Falck, J. R., Dishman, E., Karara, A. (1990) Methods Enzymol. 187, 385-394 [Medline] [Order article via Infotrieve]
  24. Capdevila, J. H., Dishman, E., Karara, A., Falck, J. R. (1991) Methods Enzymol. 206, 441-453 [Medline] [Order article via Infotrieve]
  25. Falck, J. R., Lumin, S., Blair, I., Dishman, E., Martin, M. V., Waxman, D. J., Guengerich, F. P., Capdevila, J. H. (1990) J. Biol. Chem. 265, 10244-10249 [Abstract/Free Full Text]
  26. Kuhn, H., Schewe, T., Rapoport, S. M., Brash, A. R. (1988) Basic Life Sci. 49, 945-949 [Medline] [Order article via Infotrieve]
  27. Falck, J. R., Lumin, S., Lee, S.-G., Heckmann, B., Mioskowski, C., Karara, A., Capdevila, J. H. (1992) Tetrahedron Lett. 33, 4893-4896 [CrossRef]
  28. Rossiter, B. E., Sharpless, K. B. (1984) J. Org. Chem. 49, 3707-3711
  29. Sevrioukova, I. F., Peterson, J. A. (1995) Biochimie (Paris) 77, 562-572 [CrossRef][Medline] [Order article via Infotrieve]
  30. Peterson, J. A. (1971) Arch. Biochem. Biophys. 144, 678-693
  31. Oliw, E. H. (1991) Adv. Prostaglandin Thromboxane Leukotriene Res. 21A, 197-200
  32. Karara, A., Wei, S., Spady, D., Swift, L., Capdevila, J. H., Falck, J. R. (1992) Biochem. Biophys. Res. Commun. 182, 1320-1325 [Medline] [Order article via Infotrieve]
  33. Katoh, T., Takahashi, K., Capdevila, J. H., Karara, A., Falck, J. R., Jacobson, H. R., Badr, K. F. (1991) Am. J. Physiol. 261, F578-F586 [Abstract/Free Full Text]
  34. Ho, P. P., Fulco, A. J. (1976) Biochim. Biophys. Acta 431, 249-256 [Medline] [Order article via Infotrieve]
  35. Capdevila, J. H., Wei, S., Yan, J., Karara, A., Jacobson, H. R., Falck, J. R., Guengerich, F. P., DuBois, R. N. (1992) J. Biol. Chem. 267, 21720-21726 [Abstract/Free Full Text]
  36. Capdevila, J. H., Karara, A., Waxman, D. J., Martin, M. V., Falck, J. R., Guengerich, F. P. (1990) J. Biol. Chem. 265, 10865-10871 [Abstract/Free Full Text]
  37. Karara, A., Makita, K., Jacobson, H. R., Falck, J. R., Guengerich, F. P., DuBois, R. N., Capdevila, J. H. (1993) J. Biol. Chem. 268, 13565-13570 [Abstract/Free Full Text]
  38. Groves, J. T., Han, Y.-Z. (1995) Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., eds) , 2nd Ed. , p. 3, Plenum Press, New York

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