From the Department of Chemistry, City College, City University of
New York, New York, New York 10031
 |
INTRODUCTION |
Both saturated and unsaturated fatty acids are degraded by
-oxidation. However, the breakdown of unsaturated fatty acids requires additional enzymes that specifically function in the metabolism of preexisting double bonds (1). Even-numbered double bonds
are reductively removed by the combined actions of
NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and
3,
2-enoyl-CoA isomerase (EC 5.3.3.8). In
contrast, metabolites with odd-numbered double bonds were thought to
undergo only a 3-cis to 2-trans isomerization
before reentering the
-oxidation spiral. However, recent evidence
suggests that odd-numbered double bonds can be removed by an
NADPH-dependent reduction once they are in the
5-position (2) as a result of chain shortening. A
detailed study of this conversion revealed a four-step reaction
sequence that results in a shift of the double bond from the
5- to the
2-position (3). The starting
metabolite in this reaction sequence is
2-trans-5-cis-dienoyl-CoA, which is formed from
5-cis-enoyl-CoA by acyl-CoA dehydrogenase.
2-trans-5-cis-Dienoyl-CoA can either complete the
-oxidation cycle to yield 3-cis-enoyl-CoA or can be
converted to 3,5-cis-dienoyl-CoA by enoyl-CoA
isomerase.1 A novel enzyme,
3,5,
2,4-dienoyl-CoA isomerase (4-6)
catalyzes the shift of both double bonds to produce 2-trans,
4-trans-dienoyl-CoA. The latter compound is a substrate of
2,4-dienoyl-CoA reductase and hence can be reduced by NADPH to
3-trans-enoyl-CoA, which is converted to
2-trans-enoyl-CoA by enoyl-CoA isomerase. The elucidation of
this novel pathway raised the question as to whether unsaturated fatty
acids with odd-numbered double bonds are metabolized via one or both
branches of the pathway. The two branches are referred to as the
isomerase-dependent pathway, which only requires enoyl-CoA
isomerase, and the reductase-dependent pathway which
requires dienoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, and
enoyl-CoA isomerase. Tserng and co-workers (7-9) have addressed this
question and come to the conclusion that the novel
reductase-dependent pathway is the dominant route,
especially in liver mitochondria. The goal of this study was to
elucidate the metabolic significance of the novel
reductase-dependent pathway. Answers were sought for the
following questions. Does the 5-cis double bond affect the
activities of the
-oxidation enzymes? What are the contributions of
the isomerase-dependent and the reductase-dependent pathways to the flux of unsaturated
fatty acid through the
-oxidation spiral? Does the
reductase-dependent pathway serve a special function that
cannot be assumed by the isomerase-dependent pathway?
 |
EXPERIMENTAL PROCEDURES |
Materials--
CoASH, butyryl-CoA, hexanoyl-CoA,
NAD+, NADH, NADPH, hexamethylphosphoramide,
2-mercaptoethanol, benzamidine hydrochloride, pepstatin A, acyl-CoA
oxidase from yeast, lactate dehydrogenase, and other standard
biochemicals were purchased from Sigma. Acyl-CoA oxidase from
Arthrobacter species was purchased from Boehringer Mannheim.
Bovine liver enoyl-CoA hydratase (crotonase) (10), pig heart
L-3-hydroxyacyl-CoA dehydrogenase (11), pig heart 3-ketoacyl-CoA thiolase (12), rat liver
3,
2-enoyl-CoA isomerase (13), and rat
liver
3,5,
2,4-dienoyl-CoA isomerase (4)
were purified as described. 2-trans, 4-trans-Octadienal was obtained from Bedoukian Research
(Danbury, CT). 2-trans-Octenoic acid, 2-octynoic acid, and
3-octenoic acid were purchased from Aldrich. Sep-Pak C18 cartridges
used for concentrating acyl-CoAs were purchased from Waters. The methyl
ester of 5-cis-octenoic acid was a kind gift from Dr. Howard
Sprecher (Ohio State University).
Synthesis of Substrates--
5-cis-Octenoic acid (3)
and 2-trans, 4-trans-octadienoic acid (14) were
prepared from methyl-5-cis-octenoate and 2-trans, 4-trans-octadienal, respectively, by established procedures.
5-cis-Octenoyl-CoA, 3-octenoyl-CoA, 2-octynoyl-CoA,
2-trans-octenoyl-CoA, and 2-trans, 4-trans-octadienoyl-CoA were synthesized from
5-cis-octenoic acid, 3-octenoic acid, 2-octynoic acid,
2-trans-octenoic acid, and 2-trans, 4-trans-octadienoic acid, respectively, by the mixed
anhydride method as described by Fong and Schulz (15). All acyl-CoAs
were further purified by HPLC.
For the synthesis of
2-trans-5-cis-octadienoyl-CoA, 10 µmol of
5-cis-octenoyl-CoA were incubated with 5 units of acyl-CoA oxidase from Arthrobacter species in 60 ml of 0.1 M KPi (pH 9.0) at room temperature. The
conversion of 5-cis-octenoyl-CoA to
2-trans-5-cis-octadienoyl-CoA was monitored by
analyzing aliquots of 50 µl, withdrawn from the reaction mixture at
different time intervals, by HPLC. Under the conditions described
above, 3-trans-5-cis-octadienoyl-CoA was observed
to be a minor product of the reaction. When
5-cis-octenoyl-CoA was completely converted to products,
usually after 1 h, the reaction was terminated by adjusting the pH
to 1.0 with concentrated HCl to avoid further isomerization of
2-trans-5-cis-octadienoyl-CoA to
3-trans-5-cis-octadienoyl-CoA. The protein was
removed from the reaction mixture by filtration through a 0.22-µm
pore size membrane, and the pH was readjusted to 8.0 with 4.0 N KOH. To avoid the difficult separation of small amounts
of 3,5-cis-octadienoyl-CoA from
2,5-cis-octadienoyl-CoA by HPLC, 0.5 unit of purified
dienoyl-CoA isomerase from rat liver was added to the reaction mixture
to isomerize 3,5-cis-octadienoyl-CoA to
2-trans-4-trans-octadienoyl-CoA, which can be
separated more conveniently from 2,5-cis-octadienoyl-CoA by
HPLC. After adjusting the pH to 1.0 with concentrated HCl, precipitated
protein was removed from the reaction mixture by filtering the mixture
through a 0.22-µm pore size membrane. The reaction mixture was
concentrated, after adjusting its pH to 3.0, by passing it through a
Sep-Pak mini column and eluting it with methanol. Methanol was
evaporated under a stream of N2, the resultant acyl-CoA was
redissolved in 1 ml of H2O, and the pH was adjusted to 3.0 with concentrated HCl.
2-trans-5-cis-Octadienoyl-CoA was further
purified by HPLC.
3,5-cis-Octadienoyl-CoA was synthesized by incubating 10 µmol of 5-cis-octenoyl-CoA with 5 units of acyl-CoA
oxidase from Arthrobacter species in 60 ml of 0.1 M KPi (pH 8.0) at room temperature. The
conversion of 5-cis-octenoyl-CoA to
3,5-cis-octadienoyl-CoA was monitored by analyzing aliquots
of 50 µl, taken at different time intervals from the reaction
mixture, by HPLC. When 5-cis-octenoyl-CoA was completely
converted to products, the reaction was terminated by adjusting the pH
to 1.0 with concentrated HCl, and the precipitated protein was removed
by filtration through a 0.22-µm pore size membrane after readjusting
the pH to 5.0. The resultant acyl-CoA was concentrated by use of a
SEP-Pak mini column as described above, and
3,5-cis-octadienoyl-CoA was purified by HPLC.
The synthesis of L-3-hydroxy-5-cis-octenoyl-CoA
was accomplished by incubating 10 µmol of
5-cis-octenoyl-CoA with 15 units of acyl-CoA oxidase from
yeast and 5 units of bovine liver crotonase in 60 ml of 0.1 M KPi (pH 8.0) at room temperature. The
conversion of 5-cis-octenoyl-CoA to
L-3-hydroxy-5-cis-octenoyl-CoA was monitored by
analyzing aliquots of 50 µl, withdrawn from the reaction mixture at
different time intervals, by HPLC. The reaction was terminated by
adjusting the pH to 1.0 with concentrated HCl when no further increase
in the formation of 3-hydroxy-5-cis-octenoyl-CoA was observed upon the addition of more enzymes.
For the purpose of synthesizing 3-keto-5-cis-octenoyl-CoA,
the above reaction was carried out in the presence of 10 units of pig
heart L-3-hydroxyacyl-CoA dehydrogenase, 1.0 mM
pyruvate, 1.0 mM NAD+, and 15 units of lactate
dehydrogenase. The conversion of
L-3-hydroxy-5-cis-octenoyl-CoA to
3-keto-5-cis-octenoyl-CoA was monitored by analyzing
aliquots of 50 µl, withdrawn from the reaction mixture at different
time intervals, by HPLC. The reaction was allowed to proceed for
approximately 5 h. The reaction was terminated by adjusting the pH
to 1.0 with concentrated HCl, and precipitated protein was removed by
filtration through a 0.22-µm pore size membrane. The resultant
3-keto-5-cis-octenoyl-CoA was concentrated by use of a
Sep-Pak minicolumn as described above and purified by HPLC.
3-Ketooctanoyl-CoA was synthesized by incubating 5 µmol of
2-octynoyl-CoA with 10 units of bovine liver crotonase in 5 ml of 50 mM MES (pH 6.0). The reaction was allowed to proceed for 45 min and was terminated by adjusting the pH to 1.0 with concentrated HCl. Precipitated protein was removed by filtration through a 0.22-µm
pore size membrane, and the pH was readjusted to 3.0. The product was
purified by HPLC. Concentrations of all acyl-CoA thioesters were
measured spectrophotometrically by quantifying CoASH with Ellman's
reagent (16) after cleaving the thioesters bond with NH2OH
at pH 7.0 (15).
Preparation of a Soluble Extract of Rat Liver
Mitochondria--
Rat liver mitochondria were isolated as described by
Nedergaard and Cannon (17) and stored at
70 °C. Mitochondria were precipitated by centrifugation and resuspended in an equal volume of
0.2 M KPi (pH 8.0) containing 20 mM
2-mercaptoethanol, 0.2% (v/v) hexamethylphosphoramide, 10 mM benzamidine, and pepstatin A (2 µg/ml). The
resuspended mitochondria were sonicated 5 times for 5 s each under
cooling at 4 °C and then were centrifuged at 100,000 × g for 30 min. The supernatant represented the soluble extract of mitochondria.
Enzyme and Protein Assays--
Purified crotonase from bovine
liver or crotonase present in a soluble extract of rat liver
mitochondria was assayed spectrophotometrically at 280 nm. A standard
assay mixture contained 0.2 M KPi, (pH 8.0), 20 µM 2-octenoyl-CoA or 2,5-octadienoyl-CoA, and enzyme to
give an absorbance change of approximately 0.04/min. A molar extinction coefficient of 5100 M
1 cm
1 was
used to calculate rates. Purified 3-hydroxyacyl-CoA dehydrogenase or
3-hydroxyacyl-CoA dehydrogenase present in a soluble extract from rat
liver mitochondria was assayed spectrophotometrically by measuring the
formation of NADH at 340 nm. A standard assay mixture contained 0.2 M KPi, (pH 8.0), 20 µM
2-octenoyl-CoA or 2,5-octadienoyl-CoA, 1 mM
NAD+, 0.3 mM CoASH, crotonase (0.17 unit),
3-ketoacyl-CoA thiolase (0.1 unit), and enzyme to give an absorbance
change of approximately 0.04/min. When the mitochondrial extract served
as an enzyme source, no purified thiolase was added to the assay
mixture. A molar extinction coefficient of 6220 M
1 cm
1 was used to calculate
rates. Purified 3-ketoacyl-CoA thiolase from pig heart or
3-ketoacyl-CoA thiolase present in a soluble extract from rat liver
mitochondria was assayed by measuring the formation of product by HPLC.
A standard assay contained 0.2 M KPi (pH 8.0),
0.3 mM CoASH, 20 µM 3-ketooctanoyl-CoA, or 20 µM 3-keto-5-octenoyl-CoA and enzyme to convert
approximately 20% of the substrate to product during the 1-min
incubation period. The reaction was terminated by adjusting the pH to
1.0 with concentrated HCl. After removal of precipitated protein by
filtration through 0.22-µm pore size membranes, the pH was readjusted
to 5, and the samples were analyzed by HPLC. The products were
quantified by use of standard curves established with either
hexanoyl-CoA or an equilibrium mixture of
2-trans-hexenoyl-CoA and 3-hydroxyhexanoyl-CoA. With
3-keto-5-octenoyl-CoA as the substrate, 3-cis-hexenoyl-CoA was formed when purified thiolase was used. However,
3-hydroxyhexanoyl-CoA and 2-trans-hexenoyl-CoA were the
products when the mitochondrial extract served as the enzyme source.
The thiolytic product of 3-ketooctanoyl-CoA was hexanoyl-CoA. Enoyl-CoA
isomerase was assayed spectrophotometrically with the purified enzyme
and 2,5-octadienoyl-CoA as the substrate. The increase in absorbance at
240 nm, due to the formation of 3,5-octadienoyl-CoA was recorded. An
extinction coefficient of 18,800 M
1
cm
1 was used to calculate rates. With the same substrate
but with the mitochondrial extract as an enzyme source, the increase in absorbance, due to the formation of 2,4-octadienoyl-CoA, was recorded. Purified dienoyl-CoA isomerase, which was added as a coupling enzyme in
some measurements, was found to produce maximally a 20% increase in
the rate. An extinction coefficient of 27,000 M
1 cm
1 was used to calculate
rates. With purified enoyl-CoA isomerase and 3-octenoyl-CoA as
the substrate, an increase in absorbance at 263 nm, due to the
formation of 2-enoyl-CoA, was recorded. An extinction coefficient of
6700 M
1 cm
1 was used to
calculate rates. A standard assay contained 0.2 M KPi (pH 8.0), 20 µM 2,5-octadienoyl-CoA
or 3-enoyl-CoA, and enzyme to produce an absorbance change of
approximately 0.04/min.
All spectrophotometric assays were performed an a Gilford, model 260, recording spectrophotometer at 25 °C. Kinetic parameters (apparent
Vmax and Km) were obtained by
nonlinear curve fitting using the Sigma plot program. One unit of
enzyme activity is defined as the amount of enzyme that converts 1 µmol of substrate to product per min. Protein concentrations were
determined as described by Bradford (18) with bovine serum albumin as
the standard.
Metabolic Studies--
For rate measurements, various amounts of
2,5-octadienoyl-CoA in 0.2 M KPi (pH 8.0) were
incubated with a soluble extract of rat liver mitochondria. When the
flux through the isomerase-dependent branch was evaluated,
1 mM NAD+ and 0.3 mM CoASH were
added, and the formation of NADH was recorded at 360 nm. An extinction
coefficient of 4140 M
1 cm
1 was
used to calculate rates. When rates of metabolism via the reductase-dependent pathway were determined, either no
cofactors or 1 mM NAD+, 0.3 mM
CoASH, 1 mM pyruvate, and 1 unit of lactate dehydrogenase were present. The absorbance increase at 300 nm was recorded, which
reflects the formation of 2,4-octadienoyl-CoA. An extinction coefficient of 27,000 M
1 cm
1
was used to calculate rates. The rate of 2,4-octadienoyl-CoA degradation was measured spectrophotometrically by recording the decrease of the absorbance at 300 nm due to the disappearance of the
substrate. The assay mixture contained in 0.2 M
KPi (pH 8.0), various amounts of 2,4-octadienoyl-CoA, 1 mM NAD+, 0.3 mM CoASH,
mitochondrial extract, and either 0 or 0.5 mM NADPH.
Observed absorbance changes were corrected for changes detected in the
absence of cofactors that most likely were due to the hydrolysis of the
substrate. An extinction coefficient of 27,000 M
1 cm
1 was used to calculate
rates. When the time-dependent formation of metabolites
from 2,5-octadienoyl-CoA or 3,5-octadienoyl-CoA was studied, 20 µM of either substrate in 0.2 M
KPi (pH 8.0) was incubated with 1 mM
NAD+, 0.3 mM CoASH, 0.5 mM NADPH,
and mitochondrial extract (0.1 mg/ml). Reactions were terminated after
various periods of incubation by adjusting the pH to 1 with
concentrated HCl. After readjusting the pH to 5, samples were clarified
by filtration through 0.22-µm pore size membranes and analyzed by
HPLC. Standard curves established with purified acyl-CoAs were used to
quantify the metabolites. In some experiments, a reconstituted
-oxidation system was used in place of the mitochondrial extract.
Such incubation mixtures contained in 0.2 M KPi
(pH 8.0) either 20 µM 3,5-octadienoyl-CoA or 20 µM 2,5-octadienoyl-CoA plus 1 mM
NAD+, 0.3 mM CoASH, enoyl-CoA isomerase (0.7 milliunit), enoyl-CoA hydratase (0.5 unit), 3-hydroxyacyl-CoA
dehydrogenase (18 milliunits), and 3-ketoacyl-CoA thiolase (0.11 unit).
Samples were processed and analyzed by HPLC as described above.
Analysis and Purification of Acyl-CoA Thioesters by
HPLC--
Acyl-CoA substrates and metabolites were purified and
analyzed by reverse-phase HPLC on a Waters µBondapak C18
column (30 cm × 3.9 mm) attached to a Waters gradient HPLC
system. The absorbance of the eluate was monitored at 254 nm.
Separation of metabolites was achieved by first washing the column with
50 mM ammonium phosphate (pH 5.5) containing 5% of
acetonitrile/water (9:1, v/v) for 15 min and then eluting acyl-CoAs by
linearly increasing the organic phase from 5 to 50% in 30 min at a
flow rate of 2 ml/min. When the kinetics of purified 3-ketoacyl-CoA
thiolase with 3-keto-5-octenoyl-CoA as a substrate were studied, an
isocratic elution was used for 7 min. Thereafter, a 5-50% gradient
was developed within 13 min. With 3-ketooctanoyl-CoA as substrate, the
separation of substrate and product was achieved by linearly increasing
the methanol content of the 75 mM ammonium phosphate (pH
5.5) elution buffer from 30 to 60% in 30 min at a flow rate of 2.5 ml/min. When the sample contained only acyl-CoAs, the isocratic part of
the elution program was omitted, and a linear gradient from 10 to 50%
was used to achieve elution within 30 min at a flow rate of 2 ml/min.
 |
RESULTS |
Does the 5-cis Double Bond Affect the
-Oxidation of
5-Enoyl-CoAs?--
The
-oxidation of unsaturated fatty acids with
odd-numbered double bonds yields 5-enoyl-CoA intermediates, which are
converted to 2,5-dienoyl-CoA by acyl-CoA dehydrogenase (3). The focus of this study was the mitochondrial metabolism of 2,5-octadienoyl-CoA. The degradation of 2,5-octadienoyl-CoA, which is a metabolite of
-linolenic acid, involves only soluble enzymes that are located in
the matrix of mitochondria. Hence, a soluble extract of mitochondria represents a suitable enzyme system to study the metabolism of this
compound. The direct
-oxidation of 2,5-octadienoyl-CoA requires the
sequential actions of crotonase, 3-hydroxyacyl-CoA dehydrogenase, and
3-ketoacyl-CoA thiolase (see Fig. 1,
pathway A). In an attempt to determine if the
5-cis double bond affects the rate at which 2,5-octadienoyl-CoA is metabolized via the isomerase-dependent pathway
(Fig. 1, pathway A), the apparent Km and
Vmax values were determined for these three
-oxidation enzymes using substrates with and without the
5-cis double bond. The results, shown in Table
I, demonstrate that the 5-cis
double bond affects the activities of the three enzymes differently.
With enoyl-CoA hydratase, a 3-fold higher Vmax
was observed when the substrate contained the 5-cis double
bond, while the Km was virtually unaffected. The
opposite observation was made with 3-hydroxyacyl-CoA dehydrogenase,
which yielded a 4-fold higher Km value for the
unsaturated as compared with the saturated substrate. However, the
maximal velocity of this enzyme was unaffected by the presence of the
5-cis double bond. The most adverse effect was detected with
3-ketoacyl-CoA thiolase, which exhibited with the unsaturated substrate
only one-sixth of the maximal velocity obtained with the saturated
substrate. In addition, the Km value was 5-fold
higher when the 5-cis double bond was present in the
substrate. These data demonstrate that 5-cis-enoyl-CoA intermediates can be directly metabolized by
-oxidation, but perhaps
more slowly than the corresponding saturated metabolites.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
Mitochondrial metabolism of
2-trans-5-cis-octadienoyl-CoA. A,
isomerase-dependent pathway; B,
reductase-dependent pathway; C, pathway
dependent on enoyl-CoA isomerase and dienoyl-CoA isomerase but not on
2,4-dienoyl-CoA reductase. EH, enoyl-CoA hydratase; HD, 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; C4, butyryl-CoA; C6, hexanoyl-CoA.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
The influence of a 5-cis double bond in the substrate on the kinetic
parameters of several enzymes of -oxidation
|
|
Metabolism of 2,5-Octadienoyl-CoA via the
Isomerase-dependent and Reductase-dependent
Pathways--
Since 2,5-octadienoyl-CoA can be metabolized by both the
isomerase-dependent (Fig. 1A) and
reductase-dependent (Fig. 1B) pathways, it was
important to determine the relative fluxes via these two routes in
mitochondria. For estimating the flux through the
isomerase-dependent pathway, rates of NADH formation in the
presence of NAD+ and CoASH but in the absence of NADPH were
measured spectrophotometrically with a soluble extract of rat liver
mitochondria (see Fig. 2A). Since initial velocities were determined, the simultaneous entry of
substrate into the reductase-dependent pathway was assumed to have
little or no effect on the rates of degradation via the isomerase-dependent pathway. Flux through the
reductase-dependent pathway was determined by measuring
spectrophotometrically the formation of 2,4-dienoyl-CoA at 300 nm (see
Fig. 2B) in the absence of coenzymes. A comparison of the
data shown in Fig. 2, curves A and B, indicates
that 2,5-octadienoyl-CoA is metabolized mostly (80%) by the
isomerase-dependent pathway and to a lesser extent (20%)
via the reductase-dependent route. When the rate of
2,4-dienoyl-CoA formation was measured in the presence of
NAD+, CoASH, and pyruvate plus lactate dehydrogenase to
reoxidize NADH, rates were lower by approximately 50% than those
observed in the absence of coenzymes (compare curves B and
C of Fig. 2). This decline in rates is attributed, at least
in part, to the
-oxidation of 2,4-dienoyl-CoA via route C
(see Fig. 1), which requires the involvement of enoyl-CoA isomerase and
dienoyl-CoA isomerase, but not the reductase. The direct
-oxidation
of 2,4-dienoyl-CoA, as outlined in Fig. 1C, is possible,
albeit at a low rate, because this intermediate has the
2-trans-4-trans configuration, which makes it a
substrate of the mitochondrial
-oxidation system in contrast to the
2-trans-4-cis-isomer, which is not directly
oxidized (14). The time-dependent
-oxidation of
2,5-octadienoyl-CoA by a mitochondrial extract from rat liver
mitochondria in the presence of NAD+, CoASH, and NADPH was
analyzed by HPLC (see Fig. 3). Striking was the almost instantaneous hydration of 2,5-octadienoyl-CoA to
3-hydroxy-5-octenoyl-CoA, the first metabolite of the
isomerase-dependent pathway (see Fig. 3A).
Purified enoyl-CoA hydratase was used to determine the equilibrium
ratio of 3-hydroxy-5-octenoyl-CoA to 2,5-octadienoyl-CoA. This ratio
was found to be 6.4:1. One minute into the reaction, significant
amounts of 3-keto-5-octenoyl-CoA and butyryl-CoA, the final product of
the isomerase-dependent pathway, were detected (see Fig.
3B). At the same time, only small quantities of metabolites
belonging to the reductase-dependent pathway had been
formed (marked
3,5 and
2,4 in Fig.
3B). Hexanoyl-CoA, the final product of the
reductase-dependent pathway, was detected toward the end of
the reaction, when it constituted 18% of butyryl-CoA (see Fig.
3C). Since butyryl-CoA is also formed by the direct
-oxidation of 2,4-dienoyl-CoA, approximately 20% of
2,5-octadienoyl-CoA is metabolized by the
reductase-dependent pathway. The kinetic parameters
(apparent Vmax and Km) of
several
-oxidation enzymes that are present in a soluble extract of
rat liver mitochondria were determined with substrates having 5-cis double bonds. As is apparent from the data shown in
Table II, the activity of enoyl-CoA
hydratase with 2,5-octadienoyl-CoA as a substrate is at least 50-fold
higher than the activity of the second most active enzyme in this group
of
-oxidation enzymes in rat liver mitochondria. The hydratase
activity is sufficiently high to maintain an equilibrium or a near
equilibrium situation between 2,5-octadienoyl-CoA and its product of
hydration. As a consequence, the effective concentration of
2,5-octadienoyl-CoA in the isomerization reaction was only 14% of the
expected concentration based on the flux through the pathway. Of the
first three enzymes of pathway A (see Fig. 1), the least active one was
3-hydroxyacyl-CoA dehydrogenase. However, the specific activity of this
enzyme was 6 times higher than the activity of enoyl-CoA isomerase,
which is the enzyme that determines the rate at which intermediates enter the reductase-dependent pathway (see Fig.
1B). The relative activities of the rate-determining
enzymes of the isomerase-dependent and
reductase-dependent pathway explain the observed greater
flux through the former than through the latter pathway. If pathway A
would be inhibited, then a larger percentage of 2,5-octadienoyl-CoA might enter pathway B. This hypothesis was tested by evaluating the
effect of NADH on the fluxes through pathways A and B. Since NADH is a
product inhibitor of 3-hydroxyacyl-CoA dehydrogenase, the flux through
pathway A should be inhibited, and more of the intermediate might enter
pathway B. The results shown in Fig. 4,
specifically the reduced formation of butyryl-CoA, prove the predicted
inhibition of the flux through pathway A. However, the formation of
hexanoyl-CoA via pathway B also was reduced, although only slightly, so
that almost equal amounts of butyryl-CoA and hexanoyl-CoA were formed
when either 0.5 or 1 mM NADH were present in the incubation
mixture (see Fig. 4, B and C).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Rates of 2,5-octadienoyl-CoA metabolism by a
soluble extract of rat liver mitochondria as a function of the
substrate concentration. A, rates of NADH formation in the
presence of 1 mM of NAD+ plus 0.3 mM CoASH. B, rates of 2,4-dienoyl-CoA formation
in the absence of coenzymes. C, rates of 2,4-dienoyl-CoA
formation in the presence of 1 mM NAD+, 0.3 mM CoASH, 1 mM pyruvate, and 1 unit of lactate
dehydrogenase. For experimental details, see "Experimental
Procedures."
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
HPLC analysis of metabolites formed from
2,5-octadienoyl-CoA by a soluble extract of rat liver mitochondria in
the presence of NAD+, CoASH, and NADPH. Shown are
products detected after 5 s (A), 1 min (B),
and 5 min (C). Peaks identified by use of authentic compound
were as follows: 2,5-octadienoyl-CoA ( 2,5);
3-hydroxy-5-octenoyl-CoA (3OH 5); butyryl-CoA
(C4); 3-keto-5-octenoyl-CoA (3keto- 5);
3,5-octadienoyl-CoA ( 3,5); 2,4-octadienoyl-CoA
( 2,4); and hexanoyl-CoA (C6). For details,
see "Experimental Procedures."
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Apparant kinetic constants of several -oxidation enzymes present in
a soluble extract of rat liver mitochondria with substrates having
5-cis double bonds
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Time-dependent degradation of
2,5-octadienoyl-CoA to butyryl-CoA and hexanoyl-CoA as a function of
the incubation time. Incubations contained 1 mM
NAD+, 0.5 mM NADPH, 0.3 mM CoASH,
and NADH at the following concentrations: 0 mM
(A); 0.5 mM (B); and 1 mM
(C).
|
|
Metabolism of 3,5-Octadienoyl-CoA--
The isomerization of
2,5-octadienoyl-CoA to 3,5-octadienoyl-CoA reduces the flux through the
isomerase-dependent pathway (pathway A) unless the
isomerization is freely reversible. Since the equilibrium concentration
of 3,5-octadienoyl-CoA was found to be at least 20 times higher than
the concentration of the 2,5-isomer, the rate of the
3,5
2,5 isomerization was expected to
be slow. An analysis of metabolites formed from 3,5-octadienoyl-CoA by
a soluble extract of rat liver mitochondria in the presence of
NAD+ and CoASH revealed the rapid conversion of
3,5-octadienoyl-CoA to 2,4-octadienoyl-CoA (see Fig.
5A) and the slower appearance of butyryl-CoA (see Fig. 5, B and C). This
pattern of metabolite formation is indicative of the degradation of
3,5-octadienoyl-CoA via pathway C, shown in Fig. 1. The absence of
metabolites characteristic of pathway A suggests that butyryl-CoA was
not formed via pathway A after the conversion of 3,5-octadienoyl-CoA to
2,5-octadienoyl-CoA. When NADPH was present besides NAD+
and CoASH, hexanoyl-CoA and butyryl-CoA were formed in a ratio of 3:2
(see Fig. 5D). A similar ratio was observed when the rate of
2,4-octadienoyl-CoA utilization by a soluble extract of rat liver
mitochondria was determined in the presence of either NAD+,
NADPH, and CoASH (B and C) or NAD+
and CoASH (C) (see Fig. 6).
This experiment revealed that 2,4-octadienoyl-CoA can be
-oxidized
directly (see Fig. 1, pathway C) in addition to being
degraded after the NADPH-dependent reduction of one double bond (see Fig. 1, pathway B). The fluxes through the two
pathways are of similar magnitude, with the reductive branch
facilitating a slightly faster flow than the direct oxidation. Although
the above mentioned results are indicative of the degradation of
3,5-octadienoyl-CoA via pathways B and C, a possible contribution of
pathway A cannot be excluded. The possibility of a slow degradation of
3,5-octadienoyl-CoA via pathway A (see Fig. 1) was evaluated by use of
a
-oxidation system reconstituted from purified enzymes. All soluble
-oxidation enzymes necessary to degrade 3,5-octadienoyl-CoA via
pathway A were combined at activity levels observed in a soluble
extract of rat liver mitochondria. The absence of dienoyl-CoA isomerase prevented the degradation via pathways B and C. When
2,5-octadienoyl-CoA was incubated with such a reconstituted
-oxidation system in the presence of NAD+ and CoASH, its
rapid degradation via pathway A was observed, as indicated by the
almost complete disappearance of the starting material within 1 min
(see Fig. 7A). In contrast,
3,5-octadienoyl-CoA, when incubated under identical conditions,
remained mostly unaltered after the same incubation time (see Fig.
7B). However, small amounts of butyryl-CoA and
3-keto-5-octenoyl-CoA so detected are indicative of a slow degradation
of 3,5-octadienoyl-CoA via pathway A.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
HPLC analysis of metabolites formed from
3,5-octadienoyl-CoA by a soluble extract of rat liver mitochondria in
the presence of NAD+ and CoASH. Shown are products
detected after 15 s (A), 1 min (B), and 5 min (C). D, product formed after 5 min when NADPH was present in addition to NAD+ and CoASH. Peaks identified
by use of authentic compounds were as follows: 3,5-octadienoyl-CoA
( 3,5), 2,4-octadienoyl-CoA ( 2,4),
butyryl-CoA (C4), and hexanoyl-CoA (C6). For
details, see "Experimental Procedures."
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Rates of 2,4-octadienoyl-CoA utilization by a
soluble extract of rat liver mitochondria as a function of the
substrate concentration. B + C, degradation in the presence
of NAD+, CoASH, and NADPH via pathways B and C (see Fig.
1). C, degradation in the presence of NAD+ and
CoASH via pathway C (see Fig. 1). For details, see "Experimental Procedures."
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
HPLC analysis of metabolites formed from
2,5-octadienoyl-CoA (A) and 3,5-octadienoyl-CoA
(B) by a reconstituted -oxidation system that did not
contain dienoyl-CoA isomerase or 2,4-dienoyl-CoA reductase. The
incubation time was 1 min. For details, see "Experimental
Procedures."
|
|
 |
DISCUSSION |
The recognition that 2,5-dienoyl-CoAs, which are metabolites of
unsaturated fatty acids with odd-numbered double bonds, can be degraded
by the reductase-dependent pathway (see Fig. 1B)
(3), raised the question as to whether the
isomerase-dependent pathway (see Fig. 1A) is
operative at all. If it is, the relative fluxes through both pathways
needed to be determined. Should the reductase-dependent pathway not be essential for the rapid
-oxidation of unsaturated fatty acids, then its metabolic function needs to be elucidated. The
results of this study as well as data presented by Tserng et
al. (7) demonstrate that 2,5-dienoyl-CoAs can be degraded via the
isomerase-dependent pathway. A kinetic study of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase
revealed that the presence of a 5-cis double bond in the
substrate had a positive effect on the hydration reaction but
negatively affected the dehydrogenation reaction and the thiolytic cleavage. Most severely affected by the 5-cis double bond
was 3-ketoacyl-CoA thiolase. The Vmax value
determined with 3-keto-5-octenoyl-CoA as the substrate was almost 6 times lower than the Vmax obtained with
3-ketooctanoyl-CoA. In addition, the Km for the substrate was 5 times higher when a 5-cis double bond was
present in the acyl chain. In contrast, the Vmax
of 3-hydroxyacyl-CoA dehydrogenase was unaffected by the presence of a
5-cis double bond, whereas the Km for the
unsaturated substrate was 4 times higher than the value for the
saturated substrate. Since the activity of the dehydrogenase present in
the soluble extract of rat liver mitochondria was found to be lower
than the activity of the thiolase, it seems that the 5-cis
double bond may have only a small, if any, impact on the flux of
5-cis enoyl-CoAs through this part of the
-oxidation
spiral. Moreover, it remains to be established which segment of the
total
-oxidation spiral limits the rate of fatty acid oxidation. If
the the flux through the
-oxidation pathway is not limited by the
degradation of medium chain intermediates, the lower activity of
3-hydroxyacyl-CoA dehydrogenase toward substrates with 5-cis
double bonds would not affect the rate of unsaturated fatty acid
oxidation.
Since 2,5-dienoyl-CoAs can be metabolized via the
isomerase-dependent (see Fig. 1A) and the
reductase-dependent (see Fig. 1B) pathways, it
was important to determine the contributions of both pathways to the
metabolism of 2,5-octadienoyl-CoA. The results of
concentration-dependent and time-dependent
measurements are indicative of a much slower flux through the
reductase-dependent branch (20%) than through the
isomerase-dependent branch (80%). The main reason for the
slow degradation of 2,5-octadienoyl-CoA via the
reductase-dependent pathway is the low activity of
enoyl-CoA isomerase toward 2,5-octadienoyl-CoA relative to the activity of 3-hydroxyacyl-CoA dehydrogenase with 3-hydroxy-5-octenoyl-CoA as a
substrate in rat liver mitochondria. Moreover, the steady-state concentration of 2,5-octadienoyl-CoA in an extract of rat liver mitochondria is low because of the high ratio of
[3-hydroxy-5-octenoyl-CoA]/[2,5-octadienoyl-CoA] at equilibrium
that is maintained because of the high activity of enoyl-CoA hydratase
in rat liver mitochondria. However, a restriction in the flux through
the isomerase-dependent pathway could result in an
increased entry of 2,5-octadienoyl-CoA into the
reductase-dependent pathway. This possibility was explored
by inhibiting 3-hydroxyacyl-CoA dehydrogenase with NADH and studying
the effect of this measure on product formation via both pathways. As
expected, the flux through the isomerase-dependent pathway
was reduced, but so was the degradation via the
reductase-dependent pathway. However, the fluxes through
both pathways, although reduced, were almost equal at
[NADH]/[NAD+] ratios of 0.5 and 1. Thus, the relative
contribution of the reductase-dependent pathway to the
degradation of unsaturated fatty acids with odd-numbered double bonds
appears to be more significant when the intramitochondrial
[NADH]/[NAD+] ratio is high, e.g. under
conditions of restricted energy utilization.
The results of this study do not agree with those of Tserng
et al. (9), who concluded that in liver mitochondria
5-cis-enoates are metabolized essentially by the
reductase-dependent pathway. However, different
experimental approaches were used in these two studies. Tserng et
al. analyzed intermediates of
-oxidation that were released
from rat liver mitochondria, whereas this study relied on experiments
with soluble extracts of liver mitochondria. Both experimental
approaches have their advantages and disadvantages. The advantage of
using intact mitochondria is that the intramitochondrial organization
of enzymes is maintained. However, the information provided by
intermediates released from mitochondria is difficult, if not
impossible to interpret, especially when the experiments are carried
out under conditions of restricted respiration. A major concern is the
reliance on the analysis of fatty acid oxidation intermediates that
under physiological conditions do not accumulate or accumulate only to
a very small extent (19). Experiments with mitochondrial extracts have
the advantage that coenzyme concentrations can be controlled and
concentrations of true intermediates can be measured. The disadvantage
is that the intramitochondrial organization of enzymes is lost.
However, in the absence of detailed information about the operation of
the two pathways, results obtained with mitochondrial extracts and
isolated enzymes provide the more meaningful information. Ultimately,
conclusions based on investigations with enzymes or enzyme systems will
have to be verified by use of biological systems like isolated
mitochondria or, better, intact cells that more closely reflect the
in vivo situation.
If the reductase-dependent pathway is not necessary to
ensure the efficient
-oxidation of 5-enoyl-CoAs, it may serve
another metabolic function. Experiments with 3,5-octadienoyl-CoA as a substrate indicated that this intermediate can be metabolized at a
significant rate only by its conversion to 2,4-octadienoyl-CoA. The
major reason for this situation is the greater thermodynamic stability
of 3,5-octadienoyl-CoA as compared with the 2,5-isomer. This difference
in stabilities is reflected by a better than 20:1 ratio of
[3,5-isomer]/[2,5-isomer] at equilibrium. Consequently, the reentry
of the 3,5-isomer into the isomerase-dependent pathway is
an energetically unfavorable reaction. The experiment with a system
reconstituted from
-oxidation enzymes, except for dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase, demonstrated that the metabolism of 3,5-octadienoyl-CoA via the
isomerase-dependent pathway is very slow even when this
compound is present at a high concentration. Because enoyl-CoA
isomerase is essential for the degradation of unsaturated fatty acids,
it is present in mitochondria and hence will catalyze the conversion of
2,5-dienoyl-CoAs to their 3,5-isomers. Once formed, 3,5-dienoyl-CoAs
cannot be metabolized efficiently via the
isomerase-dependent pathway. Therefore, these intermediates
must be metabolized via the reductase-dependent pathway, or
they would accumulate and thereby tie up free coenzyme A. Consequently,
the concentration of free mitochondrial coenzyme A would decline with
the result that all oxidative pathways in mitochondria would be
inhibited. Obviously, such a deleterious consequence of the unintended
2,5
3,5 isomerization must be
prevented. Since dienoyl-CoA isomerase ensures the removal of
3,5-dienoyl-CoAs, it may be present in mitochondria (4, 5) and
peroxisomes (6) as a detoxification enzyme.
An interesting finding of this study was the observation that
2,4-octadienoyl-CoA can be degraded by
-oxidation without being first reduced by 2,4-dienoyl-CoA reductase (see Fig. 1C).
This result is not completely surprising, since it had been reported that 2-trans-4-trans-decadienoyl-CoA can be
directly oxidized, whereas the
4-cis-2-trans-isomer can only be degraded after
reduction by 2,4-dienoyl-CoA reductase (14). Although the reduction of 2,4-octadienoyl-CoA is more rapid than its direct
-oxidation, both
pathways contribute significantly to the degradation of this metabolite. Hence, the term "reductase-dependent
pathway" referring to the branch initiated by enoyl-CoA isomerase is
not correct. A better term would be "dienoyl-CoA
isomerase-dependent pathway," because this enzyme is
essential for the formation of 2,4-octadienoyl-CoA. However, since the
term "reductase-dependent pathway" is simple and well
established, it will be retained with the understanding that it refers
to the
-oxidation of unsaturated fatty acid with odd-numbered double
bonds via a sequence of reactions with 2,4-dienoyl-CoA as
intermediate.