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 ( = 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withdrawn, and the organic soluble products were extracted three times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 -Detector (Radiomatic
Instruments, Tampa, FL).
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 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).
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 AcidTo a room temperature mixture of synthetic methyl
18-hydroxyeicosatetraenoate (1.5 mg) (27),
R-(+)--methoxy-
-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
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.
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,
[]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
0.26) to give the epoxy-benzoate (98 mg, 100%) as a colorless oil. 1H NMR (CDCl3,
250 MHz):
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.
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.
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.
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.
|
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 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
80 s
1) (29). More importantly, as
shown below, under the reaction conditions employed, P450BM-3 displayed
high regiochemical selectivity for the fatty acid
-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.
|
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-3To 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.)
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.
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 -2 carbon atom, with
-1
and
-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
-3 carbon
accounted for less than 1% of the total products and no metabolism was
observed at the
-1 or
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
-hydroxylases) (4), none of the
CYP4A isoforms catalyzes fatty acid or AA
-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.
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 -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
-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).
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 -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).
|
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
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
-2 sp3
carbon
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.
As mentioned, ETA lacks olefinic bonds in the vicinity of its -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.
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
-1,
-2 or
-3
sp3 carbon atoms is similar to that previously obtained
with several saturated fatty acid substrates (5, 34).
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Thus, for all the polyunsaturated fatty acids tested here, the
preferred sites for the P450BM-3 catalyzed oxygen insertion were the
-2 and
-3 carbon atoms. P450BM-3 metabolized the
-2 or the
-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.
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 -end of the AA
molecule to occupy more readily an existing active site cavity (Fig.
10). In this model, the
-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.
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.