From the Department of Chemistry, City College and Graduate School of the City University of New York, New York, New York 10031
Received for publication, September 10, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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Unsaturated fatty acids with odd-numbered double
bonds, e.g. oleic acid, can be degraded by The degradation of unsaturated and polyunsaturated fatty
acids by -oxidation
via the isomerase-dependent pathway or the
reductase-dependent pathway that differ with respect to the
metabolism of the double bond. In an attempt to elucidate the metabolic
functions of the two pathways and to determine their contributions to
the
-oxidation of unsaturated fatty acids, the degradation of
2-trans,5-cis-tetradecadienoyl-CoA, a
metabolite of oleic acid, was studied with rat heart mitochondria.
Kinetic measurements of metabolite and cofactor formation demonstrated that more than 80% of oleate
-oxidation occurs via the classical isomerase-dependent pathway whereas the more recently
discovered reductase-dependent pathway is the minor
pathway. However, the reductase-dependent pathway is
indispensable for the degradation of
3,5-cis-tetradecadienoyl-CoA, which is formed from
2-trans,5-cis-tetradecadienoyl-CoA by
3,
2-enoyl-CoA isomerase, the auxiliary
enzyme that is essential for the operation of the major pathway of
oleate
-oxidation. The degradation of
3,5-cis-tetradecadienoyl-CoA is limited by the capacity of
2,4-dienoyl-CoA reductase to reduce
2-trans,4-trans-tetradecadienoyl-CoA, which is
rapidly formed from its 3,5 isomer by
3,5,
2,4-dienoyl-CoA isomerase. It is
concluded that both pathways are essential for the degradation of
unsaturated fatty acids with odd-numbered double bonds inasmuch as the
isomerase-dependent pathway facilitates the major flux
through
-oxidation and the reductase-dependent pathway
prevents the accumulation of an otherwise undegradable metabolite.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation requires the involvement of auxiliary enzymes that act on preexisting double bonds. Even-numbered double bonds are
reductively removed via two reactions that are catalyzed by the
auxiliary enzymes 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and
3,
2-enoyl-CoA isomerase (enoyl-CoA
isomerase)1 (EC 5.3.3.8) (for
review, see Ref. 1). Odd-numbered double bonds like the
9-cis double bond, which is present in oleic acid and in
many other dietary fatty acids, are either isomerized or reduced during
-oxidation (for review, see Ref. 2). As summarized in Scheme
1, oleoyl-CoA
(I)2 is chain-shortened by
two cycles of
-oxidation to 5-cis-tetradecenoyl-CoA (II).
Dehydrogenation of the latter compound by long-chain
acyl-CoA dehydrogenase (3) produces
2-trans,5-cis-tetradecadienoyl-CoA (III), which
can complete its pass through the
-oxidation cycle. The resultant
3-cis-dodecenoyl-CoA (VI), after isomerization to 2-trans-dodecenoyl-CoA (VII), can be completely degraded via
the
-oxidation spiral. This pathway is referred to as the
isomerase-dependent pathway because it only requires
enoyl-CoA isomerase as an auxiliary enzyme. Most textbooks only mention
this pathway when they discuss the
-oxidation of oleate or
linoleate. However, the reductive removal of the double bond of
5-cis-enoyl-CoAs in mitochondria has been observed (4) and
was explained by a four-step reaction sequence (5) that would convert
2,5-tetradecadienoyl-CoA (III) to 2-tetradecenoyl-CoA (XIV). The latter
intermediate can be completely degraded via the
-oxidation spiral.
The auxiliary enzymes required for this pathway are a novel enzyme,
3,5,
2,4-dienoyl-CoA isomerase
(dienoyl-CoA isomerase) (6), in addition to 2,4-dienoyl-CoA reductase
and enoyl-CoA isomerase. This pathway has been referred to as
reductase-dependent pathway.
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Scheme 1.
-Oxidation of oleoyl-CoA in rat
mitochondria. A, isomerase-dependent pathway; B,
reductase-dependent pathway. AD, acyl-CoA dehydrogenase;
EH, enoyl-CoA hydratase; HD, L-3-hydroxyacyl-CoA
dehydrogenase; KT, 3-ketoacyl-CoA thiolase; EI,
3,
2-enoyl-CoA isomerase; DI,
3,5,
2,4-dienoyl-CoA isomerase;
DR, 2,4-dienoyl-CoA reductase.
Attempts to determine the relative fluxes through the
reductase-dependent and isomerase-dependent
pathways have not yet produced satisfactory answers for the degradation
of typical long-chain dietary fatty acids. An estimate of the
degradation of 5-cis-tetradecenoyl-CoA, an intermediate of
oleate -oxidation, via the reductase-dependent pathway
yielded values of 86% and 65% for liver and heart mitochondria, respectively (7). However, that study relied on the quantification of
fatty acid metabolites in intact mitochondria. These metabolites are
not true intermediates of
-oxidation but rather products that have
leaked from the pathway, especially when functionally compromised
mitochondria are involved (8). Hence, it is very doubtful that these
values are meaningful estimates of the flux through the
reductase-dependent pathway. In fact, when the degradation of 2-trans,5-cis-octadienoyl-CoA, a medium-chain
intermediate of linolenic acid metabolism, was studied with a soluble
extract of rat liver mitochondria in the presence of NAD+,
CoASH, and NADPH, 80% of the metabolite was observed to be degraded via the isomerase-dependent pathway (9). The uncertainty
about the contributions of the two pathways to the
-oxidation of
long-chain dietary fatty acids prompted this study of the degradation
of the oleate metabolite
2-trans,5-cis-tetradecadienoyl-CoA (III) by
solubilized rat heart mitochondria.
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EXPERIMENTAL PROCEDURES |
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Materials-- CoASH, NAD+, NADH, NADPH, dodecanoyl-CoA, decanoyl-CoA, and acetyl-CoA were purchased from Life Science Resources, Milwaukee, WI. Acyl-CoA oxidase from Arthrobacter species was bought from Roche Molecular Biochemicals. Sep-Pak C18 cartridges used for concentrating acyl-CoAs and µBondapak C18 columns (30 cm× 3.9 mm) were purchased from Waters Associates. Sigma was the supplier of most standard biochemicals. Bovine liver enoyl-CoA hydratase (crotonase) (10), recombinant pig liver L-3-hydroxyacyl-CoA dehydrogenase (11), pig heart 3-ketoacyl-CoA thiolase (12), recombinant human peroxisomal enoyl-CoA isomerase (13), rat liver enoyl-CoA isomerase (14), and recombinant rat liver dienoyl-CoA isomerase (15) were purified by published procedures. 2-trans-Dodecenoic acid was synthesized from n-decanal and malonic acid as described in principle by Linestead et al. (16). 5-cis-Tetradecenoic acid was a kind gift from Dr. Howard Sprecher, Ohio State University.
Syntheses of Substrates and
Metabolites--
5-cis-Tetradecenoyl-CoA and
2-trans-dodecenoyl-CoA were synthesized from
5-cis-tetradecenoic acid and 2-trans-dodecenoic
acid, respectively, by the mixed anhydride method as
described by Fong and Schulz (17). Both products were purified by HPLC.
For the synthesis of
2-trans-5-cis-tetradecadienoyl-CoA, a solution of 5 µmol of 5-cis-tetradecenoyl-CoA in 30 ml of 0.1 M potassium Pi (pH 9.0) was saturated with air
for 30 min and dehydrogenated by acyl-CoA oxidase at room temperature.
The near-complete conversion was achieved by the addition of 10-20
units of acyl-CoA oxidase in several aliquots over a period of 45 min.
The progress of the conversion was monitored by HPLC. When a maximal
conversion was achieved as indicated by the disappearance of
5-cis-tetradecaenoyl-CoA, the pH of the solution was
adjusted to 1.5 with 6 N HCl to terminate the reaction. Precipitated
protein was removed by filtering the solution through a 0.22-µm pore
size membrane. After adjusting the pH to 4 with 4 N KOH, the solution
was concentrated by passing it through a Sep-Pak C18
cartridge and eluting it with a small volume of methanol, which
subsequently was evaporated under reduced pressure. The resultant
2-trans-5-cis-tetradecadienoyl-CoA was purified
by HPLC. Fractions containing
2-trans-5-cis-tetradecadienoyl-CoA were combined,
concentrated as described above, and finally dissolved in deionized
water. The pH of the final preparation was adjusted to 3-4, and the
thioester concentration of this solution was determined
spectrophotometrically by quantification of CoASH with Ellman's
reagent (18) after cleaving the thioester bond with NH2OH
at pH 7.0 (17). The concentration of
2-trans,5-cis-tetradecadienoyl-CoA was calculated
by subtracting the concentration of
3,5-cis-tetradecadienoyl-CoA from that of
2-trans,5-cis-tetradecadienoyl-CoA plus
3,5-cis-tetradecadienoyl-CoA. The concentrations of
2-trans,5-cis-tetradecadienoyl-CoA plus 3,5-cis-tetradecadienoyl-CoA and of
3,5-cis-tetradecadienoyl-CoA were determined by measuring
the absorbance changes at 300 nm caused by their conversions in 0.1 M potassium Pi (pH 8.0) to 2,4-tetradecadienoyl-CoA on additions of 0.1 unit of dienoyl-CoA isomerase plus 0.05 unit of enoyl-CoA isomerase and of 0.1 unit of
dienoyl-CoA isomerase, respectively. Concentrations of 2,4-dienoyl-CoA were calculated using an extinction coefficient of 28,000 M
1·cm
1 (19).
For the synthesis of 3,5-cis-tetradecadienoyl-CoA, 5 µmol of 2-trans-5-cis-tetradecadienoyl-CoA were incubated with 10 units of human peroxisomal enoyl-CoA isomerase in 30 ml of 0.1 M potassium Pi (pH 8.0) at room temperature. The conversion was monitored by HPLC. After completion of the reaction, NAD+, CoASH, 0.2 unit of enoyl-CoA hydratase, 0.4 unit of 3-hydroxyacy-CoA dehydrogenase, and 0.4 unit of 3-ketoacyl-CoA thiolase were added to remove traces of 2-trans-5-cis-tetradecadienoyl-CoA. L-3-Hydroxy-5-cis-tetradecenoyl-CoA was prepared by incubating 2 µmol of 2-trans,5-cis-tetradecadienoyl-CoA in 10 ml of 0.1 M potassium Pi buffer (pH 8.0) with 9 units of enoyl-CoA hydratase at room temperature and separating the product from the substrate by HPLC. For the synthesis of 3-keto-5-cis-tetradecenoyl-CoA, 1.5 µmol of L-3-hydroxy-5-cis-tetradecenoyl-CoA in 10 ml of 20 mM potassium Pi buffer (pH 8.0) was incubated with 0.5 mM pyruvate and 0.5 mM NAD+ in the presence of 5 units of L-3-hydroxyacyl-CoA dehydrogenase and 7 units of lactate dehydrogenase at room temperature. The desired product was purified by HPLC. L-3-Hydroxydodecanoyl-CoA and 3-ketododecanoyl-CoA were synthesized from 2-trans-dodecenoyl-CoA and L-3-hydroxydodecanoyl-CoA, respectively, as described above for the corresponding longer chain acyl-CoAs. 2-trans,4-trans-Tetradecadienoyl-CoA was prepared by incubating 3,5-cis-tetradecadienoyl-CoA with dienoyl-CoA isomerase and purifying the product by HPLC.
Preparation of a Solubilized Extract from Rat Heart
Mitochondria--
Rat heart mitochondria were isolated as described by
Chappell and Hansford (20) and stored at 70 °C. The thawed rat
heart mitochondria were suspended in 0.2 M potassium
Pi containing 0.5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1% Triton X-100, 10 mM
benzamidine, and 5 mM 2-mercaptoethanol and incubated for 30 min on ice. The mixture was centrifuged at 100,000 × g at 4 °C for 30 min, and the supernatant was used for
metabolic assays.
Metabolic Assays--
Rates of degradation of
2-trans-5-cis-tetradecadienoyl-CoA via the
isomerase-dependent pathway were determined by incubating various amounts of the substrate in 0.2 M potassium
Pi (pH 8) with an extract of solubilized rat heart
mitochondria (0.1 mg/ml) in the presence of 1 mM
NAD+ plus 0.3 mM CoASH and measuring the rate
of NADH formation spectrophotometrically at 360 nm. An extinction
coefficient of 4,140 M1cm
1 was used to
calculate rates. A concentration of 1 mM was chosen for
NAD+ because it is saturating (21, 22), whereas 0.3 mM is the estimated concentration of free CoASH in
mitochondria at state 3 respiration with
palmitoyl-L-carnitine as substrate (23). The conversion of
2-trans-5-cis-tetradecadienoyl-CoA to
2,4-tetradecadienoyl-CoA was measured by incubating the substrate in
0.2 M potassium Pi (pH 8) with an extract of
solubilized rat heart mitochondria (0.1 mg/ml) fortified with 0.1 unit
of dienoyl-CoA isomerase. The absorbance change at 300 nm was recorded
and an extinction coefficient of 28,000 M
1·cm
1 was used to calculate rates. When
the time-dependent formation of metabolites was studied, 20 µM
2-trans-5-cis-tetradecadienoyl-CoA or 20 µM 3,5-cis-tetradecadienoyl-CoA was incubated
in 0.2 M potassium Pi (pH 8.0) with an extract
of solubilized rat heart mitochondria (0.1 mg/ml) in the presence of 1 mM NAD+, 0.3 mM CoASH, and 0.5 mM NADPH. Reactions were terminated by adjusting the pH to
1.5 with 6 N HCl. The pH was readjusted to 4.5 with 4 N KOH before the
reaction mixtures were clarified by filtration through 0.22 µm pore
size membranes and analyzed by HPLC. Extinction coefficients of 15,000, 19,650, and 28,800 M
1·cm
1 were determined
at 254 nm for acyl-CoA thioesters that have a saturated
-carbon, one
double bond, and two double bonds in conjugation with the thioester
function, respectively. These extinction coefficients were used to
calculate concentrations of metabolites from the peak areas of HPLC
chromatograms. This approach is made possible by the fact that the
total concentration of all acyl-CoA metabolites, except for newly
formed acetyl-CoA, does not change during the course of the reaction
and is equal to the starting concentration of the substrate, which was
20 µM. One unit of enzyme activity is defined as the
amount of enzyme that catalyzes the conversion of 1 µmol of substrate
to product in 1 min.
Purification and Analyses of Acyl-CoA Thioesters by
HPLC--
Acyl-CoA substrates were purified and metabolites were
analyzed by reverse-phase HPLC on a Waters µBondapak C18
column (30 cm x 3.9 mm) attached to a Waters gradient HPLC system. The
absorbance of the eluate was monitored at 254 nm. Separation of
substrates and metabolites was achieved by washing the µBondapak
C18 column with 50 mM ammonium phosphate (pH
5.5) containing 30% of acetonitrile/water (9:1, v/v) for 20 min and
then eluting acyl-CoAs by linearly increasing the organic phase from
30% to 60% in 20 min at a flow rate of 2 ml/min. All samples were
cleared of particulate matter by passing them through a 0.22-µm pore
size membrane before they were injected into the HPLC system. Diluted
samples were concentrated by passing them through Sep-Pak
C18 cartridges and eluting them with small amounts of
methanol, which subsequently were removed by evaporation under reduced pressure.
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RESULTS |
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Kinetics of 2-trans,5-cis-Tetradecadienoyl-CoA
Degradation--
2-trans,5-cis-Tetradecadienoyl-CoA
(III) is an intermediate that is formed during the -oxidation of
oleate and that can be further metabolized by either the
isomerase-dependent pathway or the
reductase-dependent pathway (see Scheme 1). The kinetics of
2-trans,5-cis-tetradecadienoyl-CoA degradation
via these two pathways were studied with rat heart mitochondria because
of their minimal contamination with peroxisomes that are estimated to
account for less than 3% of cardiac fatty acid
-oxidation (24).
Treatment of rat heart mitochondria with 1% Triton X-100 yielded a
soluble extract that contained all
-oxidation enzymes required for
the degradation of
2-trans,5-cis-tetradecadienoyl-CoA (III) to
decanoyl-CoA (X) and dodecanoyl-CoA (XVII). Because it had previously
been determined that Triton X-100 at the applied concentration did not
affect the activities of the enzymes of the
-oxidation spiral (25,
26), it was only necessary to assess how Triton X-100 affects the
activities of enoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, and
dienoyl-CoA isomerase. Such tests revealed that none of these three
auxiliary enzymes was negatively affected by 1% Triton X-100 (data not
shown). When 2-trans,5-cis-tetradecadienoyl-CoA (III) was incubated with an extract of rat heart mitochondria in the
presence of 1 mM NAD+ and 0.3 mM
CoASH but in the absence of NADPH, it was possible to determine rates
of
-oxidation via the isomerase-dependent pathway
without interference from the reductase-dependent pathway by measuring spectrophotometrically the formation of NADH at 360 nm.
The entry into the reductase-dependent pathway was
determined separately by measuring at 300 nm the accumulation of
2,4-tetradecadienoyl-CoA (XII) in the absence of any cofactor. The
results of these experiments are shown in Fig.
1. Specific activities for the
isomerase-dependent pathway are based on initial velocity
measurements that were linear during the first 2 min when, on the
average, 1.5 mol of NADH were produced per mol of degraded
2-trans,5-cis-tetradecadienoyl-CoA (III). The
conversion of 2-trans,5-cis-tetradecadienoyl-CoA
(III) to 2,4-tetradecadienoyl-CoA (XII) was measured 30 s after
initiation of the reaction, when rates were linear. When the flux of
2-trans,5-cis-tetradecadienoyl-CoA (III) through
the isomerase-dependent pathways is compared with its entry
into the reductase-dependent pathway, it is obvious that
the former pathway is dominant and that the ratio of rates for the two
pathways does not vary significantly over a considerable range of
substrate concentrations (see Fig. 1). Consequently, the results that
were obtained by studying the degradation of 2-trans,5-cis-tetradecadienoyl-CoA at one
concentration may reflect the situation in intact mitochondria, for
which the concentrations of true intermediates are unknown.
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In subsequent experiments we analyzed the time-dependent
formation of metabolites that accumulate when 20 µM
2-trans,5-cis-tetradecadienoyl-CoA (III) was
incubated with an extract of solubilized rat heart mitochondria in the
presence of 1 mM NAD+, 0.3 mM
CoASH, and 0.5 mM NADPH. Representative HPLC chromatograms are shown in Fig. 2. Product analysis
5 s after initiating the incubation revealed the rapid
hydration of
2-trans,5-cis-tetradecadienoyl-CoA (III) to
3-hydroxy-5-cis-tetradecenoyl-CoA (IV) (see Fig.
2A). Because the hydration is freely reversible, both
intermediates can enter either pathway. In addition, traces of
2-dodecenoyl-CoA (VII) and 2,4-tetradecadienoyl-CoA (XII) were
detected. These two metabolites are committed to proceed through the
isomerase-dependent pathway and
reductase-dependent pathway, respectively. After 1 min of
incubation, all intermediates of the isomerase-dependent pathway with the exception of 3-ketododecanoyl-CoA (IX) were present at
detectable levels (see Fig. 2B). Decanoyl-CoA was the final product of this metabolic sequence due to the absence of cofactors that
are necessary for its further degradation by -oxidation. Entry into
the reductase-dependent pathway had also continued as
indicated by the formation of more 2,4-tetradecadienoyl-CoA (XII),
whereas dodecanoyl-CoA (XVII) remained undetectable (see Fig.
2B). Five minutes after initiating the incubation,
dodecanoyl-CoA (XVII), the end product of the
reductase-dependent pathway under the prevailing
experimental conditions, was present together with its precursor,
2,4-tetradecadienoyl-CoA (XII) (see Fig. 2C).
3,5-Tetradecadienoyl-CoA was difficult to detect because it was
insufficiently separated from
2-trans,5-cis-tetradecadienoyl-CoA. However, it
is unlikely to accumulate because of the high activity of dienoyl-CoA
isomerase in the mitochondrial extract. After a total reaction time of
5 min, 2-trans,5-cis-tetradecadienoyl-CoA had
been completely metabolized, and all intermediates of the
isomerase-dependent pathway had been converted to
decanoyl-CoA (X). The small amount of material marked
2-C12-CoA was identified as a
nonmetabolizable side product that seems to be formed from either
2,5-tetradecadienoyl-CoA (III) or 2,4-tetradecadienoyl-CoA (XII) in a
time-dependent manner and that was eluted from the
reverse-phase column together with
2-C12-CoA.
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The kinetics of
2-trans,5-cis-tetradecadienoyl-CoA degradation
and metabolite formation are shown in Fig.
3. Most dramatic was the rapid hydration
of 2-trans,5-cis-tetradecadienoyl-CoA to
3-hydroxy-5-cis-tetradecenoyl-CoA. This reaction preceded
the slower dehydrogenation of the 3-hydroxy intermediate and the even slower appearance of the final product, decanoyl-CoA
(C10-CoA). It was noted that the utilization of
2-trans,5-cis-tetradecadienoyl-CoA and
3-hydroxy-5-cis-tetradecenoyl-CoA followed a similar time course and that the concentrations of 2-dodecenoyl-CoA and
3-hydroxydodecanoyl-CoA changed almost in parallel (see Fig. 3). Thus,
the two hydration reactions seem to be at or near equilibrium. The fact
that the levels of 3-ketoacyl-CoAs and 3-cis-dodecenoyl-CoA
were low or undetectable suggests that these intermediates are rapidly
degraded. Taken together, the observed kinetics of intermediate
formation and degradation point to the dehydrogenations of
3-hydroxyacyl-CoAs as the reactions that exert the greatest control
over the flux through the isomerase-dependent pathway. The
entry of 2-trans,5-cis-tetradecadienoyl-CoA into
the reductase-dependent pathway was initially quite rapid as indicated by the formation of 2,4-tetradienoyl-CoA but declined as
the concentration of
2-trans,5-cis-tetradecadienoyl-CoA decreased because of its hydration. However, dodecanoyl-CoA, the end product of
this pathway, was formed very slowly with the result that only a
fraction of its precursor, 2,4-tetradecadienoyl-CoA, was converted to
the final product during the five-minute incubation period, which was
sufficient for the complete conversion of all intermediates of the
isomerase-dependent pathway to the final product
decanoyl-CoA. Thus, it seems that the NADPH-dependent
reduction of 2,4-tetradecadienoyl-CoA restricts the flux through the
reductase-dependent pathway.
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Degradation of
3,5-Tetradecadienoyl-CoA--
3,5-cis-Tetradecadienoyl-CoA
is an assumed intermediate of oleate -oxidation that we did not
detect during the characterization of metabolites formed from
2-trans,5-cis-tetradecadienoyl-CoA because it was
not separated from its precursor by HPLC. In addition we asked whether
3,5-cis-tetradecadienoyl-CoA could be metabolized via the
isomerase-dependent pathway in addition to being degraded by the reductase-dependent pathway. To address these
issues, 3,5-cis-tetradecadienoyl-CoA was incubated with an
extract of rat heart mitochondria in the presence of NAD+
and CoASH, and its metabolites were analyzed by HPLC. Because the
absence of NADPH prevents degradation via the
reductase-dependent pathway, the flux through the
isomerase-dependent pathway can be evaluated. As shown in
Fig. 4A,
3,5-cis-tetradecadienoyl-CoA was rapidly converted to its
2,4 isomer, but did not enter the isomerase-dependent
pathway to a significant degree. After 5 min of incubation, a trace of
decanoyl-CoA was detected (data not shown), which could have been
formed either via the isomerase-dependent pathway as
outlined in Scheme 1 or more likely by direct
-oxidation of
2,4-tetradecadienoyl-CoA. The rapid degradation of
3,5-cis-tetradecadienoyl-CoA via the
reductase-dependent pathway was demonstrated by incubating it with an extract of rat heart mitochondria in the presence of all
required cofactors including NAD+, CoASH, and NADPH. As
shown in Fig. 4B, 3,5-cis-tetradecadienoyl-CoA was rapidly converted to its 2,4 isomer, which was slowly reduced as
indicated by the delayed appearance of dodecanoyl-CoA
(C12-CoA) in the absence of significant amounts of
downstream metabolites. This experiment demonstrates that
3,5-cis-tetradecadienoyl-CoA is only metabolized via the
reductase-dependent pathway and additionally confirms the
conclusion reached during the first part of this study that the
reduction of 2,4-tetradecadienoyl-CoA is the rate-limiting reaction in
the reductase-dependent pathway.
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Effects of NADH and Acetyl-CoA on the -Oxidation of
2-trans,5-cis-Tetradecadienoyl-CoA--
The metabolic studies
described above were carried out with NAD+, CoASH, and
NADPH as cofactors but in the absence of NADH and acetyl-CoA that are
present in mitochondria. Because NADH and acetyl-CoA may inhibit
-oxidation enzymes and thereby the flux through the pathways, we
assessed their effects on the degradation of
2-trans,5-cis-tetradecadienoyl-CoA. For this
purpose, we determined the formation of decanoyl-CoA
(C10-CoA) and dodecanoyl-CoA (C12-CoA) plus
2,4-tetradecadienoyl-CoA (
2,4-C14-CoA) as a
function of the incubation time to measure fluxes through the
isomerase-dependent pathway and
reductase-dependent pathway, respectively. Shown in Fig.
5 are the results that were obtained when
no NADH (Fig. 5A), 0.17 mM NADH (Fig.
5B), or 0.5 mM NADH (Fig. 5C) was
included in the incubation mixture in addition to the required
cofactors NAD+, CoASH, and NADPH. When the product
formation during the first 3 min was evaluated, the presence of NADH at
the lower level resulted in slightly lower rates of
-oxidation but
did not affect the relative flux through the
reductase-dependent pathway of ~10%. At the higher NADH
concentration, the rate of product formation was further reduced,
whereas the relative flux through the reductase-dependent pathway was only slightly increased to ~15% of the total. Thus, NADH
inhibits
-oxidation without significantly affecting the relative
contributions of the two pathways to the degradation of
2-trans,5-cis-tetradecadienoyl-CoA. The effect of
acetyl-CoA on the operation of the two pathways was also investigated.
An increasing substitution of up to 80% of CoASH in the incubation mixture by acetyl-CoA did not affect the rate of
2-trans,5-cis-tetradecadienoyl-CoA
-oxidation,
nor did it change the contributions of the two pathways to this process
(data not shown).
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DISCUSSION |
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The demonstration that unsaturated fatty acids with odd-numbered
double bonds can be degraded by two pathways, the
isomerase-dependent pathway and the
reductase-dependent pathway (5), prompted the questions as
to how much each pathway may contribute to the total flux through
-oxidation and what specific functions the two pathways may have.
The focus of this investigation was the
-oxidation of unsaturated
dietary fatty acids in mitochondria. Given that oleic acid is
abundantly present in the human diet and only contains a
9-cis double bond, it was selected as the fatty acid best
suited for this study. The oleate metabolite,
2-trans,5-cis-tetradecadienoyl-CoA, served as the substrate for the required flux measurements and product
determinations. Mitochondria from rat heart were used because of their
minimal contamination by peroxisomes, which contain a
-oxidation
system different from the mitochondrial one. Mitochondria were
solubilized with Triton X-100 to obtain a system that, in contrast to
intact mitochondria, would permit rate measurements of the individual
pathways. The concentration of CoASH was fixed at 0.3 mM
because this is its estimated concentration in mitochondria that
rapidly oxidize fatty acids (23). The concentrations of NAD+ and NADPH were set at 1 mM and 0.5 mM, respectively, because these are saturating
concentrations even though they are lower than their estimated
intramitochondrial concentrations. When rates of
2-trans,5-cis-tetradecadienoyl-CoA degradation
via the isomerase-dependent pathway were compared with
rates of its entry into the reductase-dependent pathway,
the former pathway was estimated to account for more than 85% of the
-oxidation of this metabolite of oleic acid. Similar results were
obtained when the accumulation of products was determined.
Decanoyl-CoA, which is formed via the isomerase-dependent pathway, accounted for 85% of the products formed from
2-trans,5-cis-tetradecadienoyl-CoA. The ratio of
substrate utilization via the two pathways varied little over a
significant range of
2-trans,5-cis-tetradecadienoyl-CoA concentrations
and hence may reflect the relative contributions of the two pathways to
-oxidation under a variety of conditions, including conditions that
exist in intact mitochondria. However, intramitochondrial conditions
may change during
-oxidation, especially as NAD+ is
converted to NADH and CoASH to acetyl-CoA. Increased concentrations of
NADH and acetyl-CoA may inhibit
-oxidation and thereby affect the
relative contributions of the two pathways. This idea was tested by
determining the effects that NADH and acetyl-CoA have on the formation
of products via the two pathways. When 15% of the total NADH was in
the reduced form, the relative contributions of the two pathways were
unchanged, even though the total flux through
-oxidation was
reduced. An increase of NADH to one-third of the total coenzyme level
further reduced the rate of oxidation but only slightly increased the
relative contribution of the reductase-dependent pathway
from 10% to 15%. Because only 5% of the total NAD+ is
estimated to be in the reduced state during fatty acid
-oxidation in
actively respiring mitochondria (23), it is unlikely that NADH would
significantly change the contribution of the
reductase-dependent pathway to oleate
-oxidation. The
same conclusion was reached with regard to the effect of acetyl-CoA.
This product of
-oxidation affected neither the rate of the process
nor the contributions of the two pathways even when it comprised 80%
of the total CoA content of the system. A major reason for the limited
flux through the reductase-dependent pathway is the rapid
and dramatic decrease in the concentration of
2-trans,5-cis-tetradecadienoyl-CoA due to its
hydration. The consequence is a greatly reduced rate of its
isomerization to 3,5-cis-tetradecadienoyl-CoA, the first
metabolite of the reductase-dependent pathway. Together,
the results of this study lead to the conclusion that the
reductase-dependent pathway only makes a minor contribution
to the total
-oxidation of oleate.
If the reductase-dependent pathway contributes little to
the -oxidation of oleate, what is its metabolic function? In an attempt to answer this question, we studied the degradation of 3,5-cis-tetradecadienoyl-CoA, the first metabolite of oleate
with two conjugated double bonds. Although it was assumed that this oleate intermediate could be metabolized via the
reductase-dependent pathway, it was uncertain whether it
also could be degraded by way of the isomerase-dependent
pathway. The results clearly demonstrate that
3,5-cis-tetradecadienoyl-CoA is rapidly converted to
2,4-tetradecadienoyl-CoA, which is reduced by
NADPH-dependent 2,4-dienoyl-CoA reductase before being
degraded by
-oxidation to dodecanoyl-CoA. Because only a trace of
decanoyl-CoA was detected, 3,5-cis-tetradecadienoyl-CoA is
not a substrate of the isomerase-dependent pathway nor is
its product, 2,4-tetradecadienoyl-CoA, effectively degraded by direct
-oxidation. The first observation agrees with the previous
conclusion that 3,5-dienoyl-CoAs cannot be metabolized via the
isomerase-dependent pathway (9). This is most likely due to
the unfavorable energetics of the 3,5-dienoyl-CoA to 2,5-dienoyl-CoA
conversion. Surprising was the observation that
2-trans,4-trans-tetradecadienoyl-CoA, in contrast
to the medium-chain metabolite
2-trans,4-trans-octadienoyl-CoA (9), was not
directly degraded by
-oxidation. It should be noted that
2-trans,4-trans-decadienoyl-CoA but not its
2-trans,4-cis isomer is a substrate, albeit a
poor one, of direct
-oxidation (19). The most likely reason for the
different reactivities of 2,4-tetradecadienoyl-CoA and
2,4-octadienoyl-CoA is the involvement of two different sets of
-oxidation enzymes. 2,4-Octadienoyl-CoA is presumably hydrated by
crotonase and the resultant 3-hydroxyoctanoyl-CoA is dehydrogenated by
3-hydroxyacyl-CoA dehydrogenase, because both of these enzymes are more
active with short-chain and medium-chain substrates than with
long-chain ones (17, 21, 22). In contrast, 2,4-tetradecadienoyl-CoA
would most likely be acted upon by long-chain enoyl-CoA hydratase and
long-chain 3-hydroxyacyl-CoA dehydrogenase of the trifunctional
-oxidation complex because crotonase and 3-hydroxyacyl-CoA
dehydrogenase exhibit little activity toward substrates with acyl
chains having 14 carbon atoms (22). Because the equilibrium
concentration of 3-hydroxy-4-enoyl-CoA formed by hydration of
2,4-dienoyl-CoAs is extremely low (equilibrium constant for the
hydration is 0.003) (27), the 3-hydroxy intermediate would only be
dehydrogenated at a measurable rate if the catalytic efficiency and
concentration of the relevant 3-hydroxyacyl-CoA dehydrogenase are
sufficiently high. These conditions seem to be met for the matrix
3-hydroxyacyl-CoA dehydrogenase acting on 3-hydroxyoct-4-enoyl-CoA but
not for long-chain 3-hydroxyacyl-CoA dehydrogenase catalyzing the
dehydrogenation of 3-hydroxy-tetradec-4-enoyl-CoA. The general
conclusion is that once 3,5-cis-tetradecadienoyl-CoA has
been formed, it can be effectively degraded only via the
reductase-dependent pathway. In the absence of this
pathway, 3,5-dienoyl-CoAs would most likely accumulate and impair the
oxidative function of mitochondria because of a decline of free CoA and
possibly by inhibiting some of the enzymes of
-oxidation.
The reductase-dependent pathway is, however, the major
pathway for the -oxidation of unsaturated fatty acids with
conjugated double bonds. Such fatty acids, specifically conjugated
linoleic acid, are constituents of the human diet because they are
formed in ruminants and during the partial hydrogenation of fats. The most common conjugated linoleic acid is
9-cis,11-trans-octadecadienoic acid.
-Oxidation of this fatty acid is expected to produce
3-cis,5-trans-dodecadienoyl-CoA as an
intermediate, which can only be degraded by way of the
reductase-dependent pathway. As previously pointed out
(28),
-oxidation of
9-cis,11-trans-octadecatrienoic acid also yields
2-trans,5-cis,7-trans-tetradecatrienoyl-CoA
as an intermediate. This metabolite might be degraded in part via the reductase-dependent pathway that requires the
participation of
3,5,7,
2,4,6-trienoyl-CoA
isomerase, which is an inherent activity of dienoyl-CoA isomerase
(15).
Surprising and interesting was the observed accumulation of
2,4-tetradecadienoyl-CoA during the -oxidation of either
2-trans,5-cis-tetradecadienoyl-CoA or
3,5-cis-tetradecadienoyl-CoA. This finding prompted the idea that the reaction catalyzed by 2,4-dienoyl-CoA reductase may limit the
flux through the pathway even though the entry into this pathway is
already restricted by competition with the dominant
isomerase-dependent pathway. A previous evaluation of a
possible control exerted by 2,4-dienoyl-CoA reductase over the
-oxidation of oleic acid and docosahexaenoic acid in cardiomyocytes
came to the conclusion that an increase in the activity of
2,4-dienoyl-CoA reductase in response to the treatment of rats with
growth hormone did not result in higher rates of
-oxidation (29).
It is possible, however, that isolated and mostly quiescent
cardiomyocytes are not suitable for such study because their low
energy need severely restricts fatty acid oxidation, with the possible
result that none of the reactions of
-oxidation is limiting the rate
of the energy production.
In summary, the reductase-dependent pathway only makes a
minor contribution to the -oxidation of oleic acid, which is mostly degraded via the classical isomerase-dependent pathway.
However, the reductase-dependent pathway is essential for
the degradation of 3,5-cis-tetradecadienboyl-CoA, which is
formed from the oleate metabolite
2-trans,5-cis-teradecadienoyl-CoA by enoyl-CoA
isomerase that functions in the isomerase-dependent
pathway. The reductase-dependent pathway is also essential
for the
-oxidation of conjugated linoleic acid like
9-cis,10-trans-octadecadienoic acid.
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FOOTNOTES |
---|
* This work was supported by U. S. Public Health Service Grant HL30847 from the National Heart, Lung, and Blood Institute and by Grant RR03060 to Research Centers of Minority Institutions.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 Chemistry,
City College of CUNY, Convent Ave. at 138th St., New York, NY 10031. Tel.: 212-650-8323; Fax: 212-650-8322; E-mail:
hoschu@sci.ccny.cuny.edu.
Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M209261200
2 The roman numerals refer to the structures of oleate metabolites presented in Scheme 1.
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ABBREVIATIONS |
---|
The abbreviations used are:
enoyl-CoA isomerase, 3,
2-enoyl-CoA isomerase;
dienoyl-CoA
isomerase,
3,5,
2,4-dienoyl-CoA
isomerase.
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