From the Department of Chemistry, City College, City University of New York, New York, New York 10031
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mitochondrial metabolism of unsaturated fatty
acids with conjugated double bonds at odd-numbered positions,
e.g. 9-cis,11-trans-octadecadienoic acid, was investigated. These fatty acids are substrates of
The The Since polyunsaturated fatty acids with conjugated double bonds (such
as, for example, conjugated linoleic acid) are formed during the
partial catalytic hydrogenation of fats (3) and in ruminants (4), they
are constituents of the human diet, and hence, their degradation by
Materials--
CoASH, Q-Sepharose, polybuffer exchanger 94, polybuffer 96, reactive red 120, Sepharose CL-6B, phenylmethylsulfonyl
fluoride, polyethylene glycol with an average Mr
of 8000, benzamidine hydrochloride, acyl-CoA oxidase from
Arthrobacter species, Staphylococcus aureus (Cowan strain) suspension (10%, w/v), and all standard
biochemicals were obtained from Sigma.
(4-Carboxybutyl)triphenylphosphonium bromide,
trans-2-pentenal, and lithium bis(trimethylsilyl)amide were
purchased from Aldrich. Hydroxylapatite, the dye reagent for protein
assays, and materials for immunoblotting, including alkaline
phosphatase-conjugated goat anti-rabbit IgG, were bought from Bio-Rad.
The multifunctional protein I from rat liver peroxisomes (5, 6),
enoyl-CoA hydratase (crotonase) from bovine liver (7), enoyl-CoA
isomerase from rat liver (8), L-3-hydroxyacyl-CoA dehydrogenase from pig heart (9), and dienoyl-CoA isomerase from rat
liver (10) were purified as described. Methyl
5-cis-octenoate (generously provided by Dr. H. Sprecher,
Ohio State University) was saponified as described (11) to yield
5-cis-octenoic acid. The CoA thioesters of
5-cis-octenoic acid and 5,7-decadienoic acid were
synthesized by the mixed anhydride method as described by Fong and
Schulz (12) and purified by
HPLC.1 3,5-Octadienoyl-CoA
and 3,5,7-decatrienoyl-CoA were prepared by the combined actions of
acyl-CoA oxidase and peroxisomal multifunctional protein I as described
by Luo et al. (10). 2,5,7-Decatrienol-CoA was prepared by
dehydrogenating 5,7-decadienoyl-CoA with acyl-CoA oxidase at pH 9 (13).
Concentrations of thioesters were determined by measuring CoASH
according to Ellman (14) after cleaving the thioester bond with 1 M NH2OH at pH 7.0 (12).
Synthesis of 5,7-Decadienoic Acid--
5,7-Decadienoic acid was
synthesized by a Wittig reaction using the procedure of Maryanoff
et al. (15). Twenty mmol of
(4-carboxybutyl)triphenylphosphonium bromide in 10 ml of anhydrous
tetrahydrofuran were combined with 42 mmol of lithium
bis(trimethylsilyl)amide in 42 ml of tetrahydrofuran at 25 °C and
under N2 with stirring. After 15 min, 16 mmol of trans-2-pentenal in 10 ml of tetrahydrofuran were added
slowly. The color of the reaction changed from red to yellow. After 60 min, 50 ml of water were added to quench the reaction. The mixture was
extracted with ether to remove the unreacted aldehyde. The aqueous
solution was acidified with 10% HCl and extracted with ether. The
combined ether extracts were extracted with water, dried over anhydrous
Na2SO4, and concentrated to yield 0.82 g of 5,7-decadienoic acid (~30% yield). The structure of the product was confirmed by mass spectrometry, which showed the expected [M + NH4+] peak at 186.
Enzyme and Protein Assays--
Dienoyl-CoA isomerase was assayed
spectrophotometrically by measuring the increase in absorbance at 300 nm on a Hitachi Model U-3000 spectrophotometer at 25 °C as described
by Luo et al. (10). A typical assay mixture contained 20 µM 3,5-octadienoyl-CoA in 0.2 M potassium
Pi (pH 8.0). An extinction coefficient of 27,800 M Purification of Trienoyl-CoA Isomerase from Pig
Heart--
Trienoyl-CoA isomerase was purified by the procedure
developed for the purification of dienoyl-CoA isomerase (10). All
operations were carried out at 4 °C. One frozen pig heart (330 g)
was minced and blended with 1 liter of 20 mM potassium
Pi (pH 8.3) containing 5 mM mercaptoethanol, 1 mM EDTA, 1 mM EGTA, 1 mM
benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride
(buffer A). The resulting suspension was centrifuged at 6500 × g for 20 min. Polyethylene glycol was added to the
supernatant to achieve a final concentration of 10%. After keeping the
suspension for 30 min, it was centrifuged at 6500 × g
for 20 min. The pellet was resuspended in 200 ml of buffer A containing
0.2 M KCl. After stirring it overnight, the suspension was
centrifuged at 100,000 × g for 1 h. The
supernatant was diluted with 4 volumes of buffer A to lower the salt
concentration and was applied to a Q-Sepharose column (2.5 × 50 cm) previously equilibrated with buffer A. The column was extensively
washed with buffer A and then eluted with 600 ml of buffer A containing 0.2 M KCl. Fractions with trienoyl-CoA isomerase activity
were combined, and the pH was adjusted to 6.3 with 1 M
KH2PO4. The mixture was applied to a
hydroxylapatite column (2.5 × 25 cm) equilibrated with 5 mM potassium Pi (pH 6.3) containing 5 mM mercaptoethanol, 1 mM EDTA, 1 mM
EGTA, 1 mM benzamidine, and 0.5 mM
phenylmethylsulfonyl fluoride (buffer B). The column was washed with
buffer B containing 0.5 M KCl and then was developed with a
gradient made up of 300 ml of buffer B and 300 ml of buffer B
containing 0.8 M potassium Pi (pH 6.3).
Fractions of 6 ml were collected, and the active fractions were
combined and concentrated in an Amicon concentrator with a YM-10
membrane. After dialyzing overnight against 25 mM ethanolamine/acetic acid (pH 9.4) containing 5 mM
mercaptoethanol, 1 mM EDTA, 1 mM benzamidine,
0.5 mM phenylmethylsulfonyl fluoride, and 20% glycerol
(buffer C), the sample was applied to a chromatofocusing column
(1.5 × 25 cm) containing polybuffer exchanger 94 equilibrated with buffer C. The column was washed with buffer C and then developed with 12 column volumes of polybuffer 96 adjusted to pH 6.0 with acetic
acid. Fractions of 5 ml were collected, and the active fractions were
combined and concentrated. Thereafter, the sample was passed through a
Sepharose CL-6B column (1.5 × 90 cm) equilibrated with 10 mM potassium Pi (pH 7.0) containing 1 mM EDTA, 5 mM mercaptoethanol, and 25%
glycerol (buffer D). The active fractions were combined and loaded onto
a reactive red 120 column (1.5 × 13 cm) previously equilibrated
with buffer D. After washing with buffer D, the column was eluted with
15 µM
2-trans,4-trans-decadienoyl-CoA. Fractions of 2 ml were collected and assayed for both dienoyl-CoA and trienoyl-CoA isomerase activities. Active fractions were analyzed by
SDS-polyacrylamide gel electrophoresis and stained with Coomassie
Brilliant Blue. The intensities of the bands were determined by gel
scanning with a densitometer.
Isolation of Mitochondria and Respiration Measurements--
Rat
liver mitochondria were isolated as described by Nedergaard and Cannon
(17). For respiration measurements, 1.5 mg of rat liver mitochondria
were incubated in 1.9 ml of a basal medium containing 20 mM
Tris-HCl (pH 7.4), 4 mM potassium Pi, 0.1 M KCl, 4 mM MgCl2, and 0.1 mM EGTA. To this mixture were added, in the indicated
orders, bovine serum albumin (0.5 mg/ml), 0.5 mM
L-carnitine, 0.5 mM L-malate, 1 mM ADP, and one of the following: 15 µM
linoleoyl-CoA, 15 µM
9-cis,11-trans-octadienoyl-CoA, 0.1 mM decanoic acid, or 0.1 mM 5,7-decadienoic
acid. Rates of respiration were measured polarographically with a
Clarke-type oxygen electrode attached to a Yellow Springs-oxygraph.
Metabolism of 2,5,7-Decatrienoyl-CoA--
The direct
Immunoprecipitation--
Dienoyl-CoA isomerase (2 µg) in 50 mM potassium Pi (pH 7) containing 1 mM benzamidine, 1 mM EDTA, and 5 mM
mercaptoethanol was combined with various amounts of anti-dienoyl-CoA
isomerase serum containing between 0 and 300 µg of protein. The total
volume was 0.2 ml. This mixture was kept for 20 min at 25 °C and
then combined with 0.2 ml of a suspension containing 10% (w/v)
S. aureus (Cowan strain). After an additional 10 min of
incubation at 25 °C, the mixture was centrifuged at 13,000 × g for 3 min. The supernatant was transferred to clean tubes
and kept at 4 °C until assayed for dienoyl-CoA and trienoyl-CoA
isomerase activities.
Analysis and Purification of Acyl-CoA Thioesters by
HPLC--
Acyl-CoAs were purified or 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 effluent was monitored at 254 nm. Separation of different acyl-CoA thioesters was
achieved by linearly increasing the acetonitrile/H2O (9:1, v/v) content of the 50 mM ammonium phosphate buffer (pH
5.5) from 20 to 55% at a flow rate of 2 ml/min. When acyl-CoAs were
purified, the desired fraction of the effluent was collected. After
evaporation of acetonitrile under vacuum, the product was concentrated
by use of a Sep-Pak C18 cartridge.
Metabolism of 5,7-Decadienoyl-CoA--
The
In a preliminary experiment, the capacity of mitochondria to oxidize
fatty acids with odd-numbered conjugated double bonds was assessed. The
data presented in Table I demonstrate
that such fatty acids supported the respiration of coupled rat liver mitochondria at rates that were slightly lower than those obtained with
the corresponding fatty acids having either non-conjugated double bonds
or no double bond at all. This result suggests the presence of a
mitochondrial pathway for the
The step-by-step degradation of 5,7-decadienoyl-CoA was studied
spectrophotometrically by use of purified enzymes. The spectrum of
5,7-decadienoyl-CoA (shown in Fig.
2A, spectrum 1) is
characterized by a major absorbance band centered around 230 nm and a
shoulder at 260 nm. These absorbances are attributed to the diene and
CoA chromophores, respectively. Treatment of 5,7-decadienoyl-CoA with acyl-CoA oxidase produced a 40% increase in the absorbance at 263 nm
(Fig. 2A, compare spectra 3 and 1).
Such a change in absorbance agrees with the conversion of an acyl-CoA
to 2-trans-enoyl-CoA. 5,7-Decadienoyl-CoA is also a
substrate of medium-chain acyl-CoA dehydrogenase. In fact, it is a
better substrate than is decanoyl-CoA (0.66 versus 0.45 units/mg). When 2,5,7-decatrienoyl-CoA was treated with enoyl-CoA
isomerase, the absorbance at 260 nm increased, whereas the absorbance
around 230 nm decreased (Fig. 2A). These absorbance changes
agree with the isomerization of a conjugated diene to a conjugated
triene that would take place during the conversion of
2,5,7-decatrienoyl-CoA to 3,5,7-decatrienoyl-CoA. The same absorbance
changes were observed when 5,7-decadienoyl-CoA was reacted with
acyl-CoA oxidase and peroxisomal multifunctional protein I, which
harbors enoyl-CoA isomerase activity (Fig. 2B).
When the suspected 3,5,7-decatrienoyl-CoA was treated with a soluble
extract of rat liver mitochondria or a partially purified preparation
of dienoyl-CoA isomerase, an absorbance band close to 340 nm appeared,
whereas the absorbance at 260 nm declined (Fig. 2C). The
increase in the absorbance at 340 nm agrees with the formation of a
2,4,6-trienoyl-CoA, which was reported to have an absorbance maximum at
337 nm (2). Since 2,4,6-octatrienoyl-CoA was reported to be reduced by
NADPH-dependent 2,4-dienoyl-CoA reductase (2), this
reaction was used to confirm the structure of the suspected
2,4,6-decatrienoyl-CoA (Fig. 2, C, spectrum 5; and D, spectrum 1). As shown in Fig.
2D, the chromophore at 340 nm disappeared in a
time-dependent manner when NADPH and 2,4-dienoyl-CoA reductase were added to the buffered solution of
2,4,6-decatrienoyl-CoA.
Further evidence for the identity of 2,4,6-decatrienoyl-CoA was
obtained by its enzymatic conversion and HPLC analysis of the resultant
products. For this purpose, synthetic 5,7-decadienoyl-CoA (Fig.
1A) was converted to 3,5,7-decatrienoyl-CoA by acyl-CoA oxidase and peroxisomal multifunctional protein I. The resultant two
compounds (Fig. 1B) are assumed to be the
3-cis-isomer (minor peak) and 3-trans-isomer
(major peak) of 3,5,7-decatrienoyl-CoA because both are converted to
2,4,6-decatrienoyl-CoA by a partially purified preparation of
dienoyl-CoA isomerase exhibiting trienoyl-CoA isomerase activity (Fig.
1C). The reduction of 2,4,6-decatrienoyl-CoA (Fig.
1D) by NADPH in the presence of 2,4-dienoyl-CoA reductase from Escherichia coli yielded a single product that was
eluted at the same position as was the starting material (Fig. 1,
compare D and E). However, the addition of
crotonase to the reaction product, presumed to be 2,6-decadienoyl-CoA
because the E. coli reductase catalyzes the reduction of the
4,5-double bond, produced a more polar compound, most likely
3-hydroxydec-6-enoyl-CoA (Fig. 1F). In contrast, the
addition of crotonase to 2,4,6-decatrienoyl-CoA (Fig. 1D)
did not produce a product of different polarity. This experiment
supports the assigned structures of 2,4,6-decatrienoyl-CoA (Fig.
1D) and 2,6-decadienoyl-CoA (Fig. 1E) because
2-enoyl-CoA compounds are hydrated to a significant extent only when
the 2-double bond is in conjugation with the thioester group, but not
when it is part of a more extended chromophore (18).
Although 5,7-decadienoyl-CoA can be converted enzymatically to
2,4,6-decatrienoyl-CoA and further degraded after the
NADPH-dependent reduction of the latter intermediate, the
operation of this pathway in mitochondria had not been demonstrated.
Toward this end, the metabolism of 2,5,7-decatrienoyl-CoA by a soluble
extract of rat liver mitochondria was investigated. Rates of the direct
Identification and Characterization of Trienoyl-CoA
Isomerase--
The identification of trienoyl-CoA isomerase prompted
its further characterization and purification. Since a partially
purified preparation of dienoyl-CoA isomerase exhibited trienoyl-CoA
isomerase activity, the relationship between these two enzyme
activities was evaluated by an immunoprecipitation experiment. As is
apparent from Fig. 4, the trienoyl-CoA
isomerase activity was precipitated by antibodies raised against
dienoyl-CoA isomerase. The two immunoprecipitation curves were close
enough to suspect that the two enzymes might be associated with the
same protein.
The relationship between trienoyl-CoA and dienoyl-CoA isomerases was
further investigated by studying their behaviors during purification.
The enzymes were purified from pig heart to minimize a possible
interference by peroxisomal forms of these enzymes. The results of this
purification effort, summarized in Table
II, demonstrate the co-purification of
the two enzymes. The activities of trienoyl-CoA and dienoyl-CoA
isomerases remained inseparable throughout the procedure even though
the dienoyl-CoA isomerase/trienoyl-CoA isomerase ratio changed from
35:1 to 143:1. The result of the last purification step, the elution of
the purified enzyme from a reactive red 120 column, is shown in Fig.
5A. The elution of trienoyl-CoA isomerase from this column coincided with the appearance of dienoyl-CoA isomerase and was proportional to the amount of protein
present in each fraction. Moreover, an analysis of individual column
fractions by SDS-polyacrylamide gel electrophoresis revealed the
presence of only one band (Fig. 5B) that corresponded to a protein with a molecular mass close to 32 kDa, which is the mass reported for dienoyl-CoA isomerase.
The observation that the CoA derivatives of fatty acids with two
conjugated double bonds at odd-numbered positions, e.g.
9-cis,11-trans-octadecadienoyl-CoA (Fig.
6, compound I), sustain
mitochondrial respiration demonstrated their degradation by
mitochondrial -oxidation in isolated rat liver mitochondria and hence are expected
to yield 5,7-dienoyl-CoA intermediates. 5,7-Decadienoyl-CoA was used to study the degradation of these intermediates. After introduction of a
2-trans-double bond by acyl-CoA dehydrogenase or acyl-CoA oxidase, the resultant 2,5,7-decatrienoyl-CoA can either continue its
pass through the
-oxidation cycle or be converted by
3,
2-enoyl-CoA isomerase to
3,5,7-decatrienoyl-CoA. The latter compound was isomerized by a novel
enzyme, named
3,5,7,
2,4,6-trienoyl-CoA
isomerase, to 2,4,6-decatrienoyl-CoA, which is a substrate of
2,4-dienoyl-CoA reductase (Wang, H.-Y. and Schulz, H. (1989)
Biochem. J. 264, 47-52) and hence can be completely degraded via
-oxidation.
3,5,7,
2,4,6-Trienoyl-CoA isomerase was
purified from pig heart to apparent homogeneity and found to be a
component enzyme of
3,5,
2,4-dienoyl-CoA
isomerase. Although the direct
-oxidation of 2,5,7-decatrienoyl-CoA seems to be the major pathway, the degradation via 2,4,6-trienoyl-CoA makes a significant contribution to the total
-oxidation of this intermediate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation of typical polyunsaturated fatty acids requires
the involvement of three auxiliary enzymes in addition to the enzymes
that catalyze the four basic reactions of the
-oxidation spiral (1).
The auxiliary enzymes are
3,
2-enoyl-CoA
isomerase
(
3-cis-
2-trans-enoyl-CoA
isomerase, EC 5.3.3.8; referred to hereafter as enoyl-CoA isomerase),
2,4-dienoyl-CoA reductase (4-enoyl-CoA reductase (NADPH), EC 1.3.1.34),
and
3,5,
2,4-dienoyl-CoA isomerase
(referred to hereafter as dienoyl-CoA isomerase). These enzymes
catalyze either the reduction or isomerization of double bonds once the
double bonds are close to the thioester function as the result of chain
shortening. Consequently, double bonds either are reductively removed
or are shifted to yield 2-trans-enoyl-CoAs, which are
intermediates of the
-oxidation spiral. Polyunsaturated fatty acid
with conjugated double bonds may yield intermediates with more extended
chromophores. For example, a fatty acid with two conjugated double
bonds at even-numbered positions is assumed to be chain-shortened by
-oxidation to 4,6-dienoyl-CoA and is then converted to
2,4,6-trienoyl-CoA by acyl-CoA dehydrogenase. The further metabolism of
this intermediate is facilitated by 2,4-dienoyl-CoA reductase, which
catalyzes the reduction of one double bond of the 2,4,6-trienoyl-CoA
chromophore to yield 3,6-dienoyl-CoA (2).
-oxidation of a fatty acid with two conjugated double bonds at
odd-numbered positions would produce 5,7-dienoyl-CoA, which may be
dehydrogenated by acyl-CoA dehydrogenase to 2,5,7-trienoyl-CoA. The
latter compound may be chain-shortened to 3,5-dienoyl-CoA or isomerized
to 3,5,7-trienoyl-CoA by enoyl-CoA isomerase. The further metabolism of
3,5,7-trienoyl-CoA would require the action of a
3,5,7,
2,4,6-trienoyl-CoA isomerase. Such
an enzyme has not been described so far.
-oxidation deserves to be studied. It was the aim of this study to
elucidate the further metabolism of 5,7-dienoyl-CoA and to identify,
isolate, and characterize a suspected
3,5,7,
2,4,6-trienoyl-CoA isomerase
(referred to hereafter as trienoyl-CoA isomerase).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm
1 was used to calculate
rates. Trienoyl-CoA isomerase activities were determined by monitoring
the absorbance increase at 337 nm. An extinction coefficient of 49,300 M
1 cm
1 was used to calculate
rates (2). A typical assay mixture contained 20 µM
3,5,7-decatrienoyl-CoA in 0.2 M potassium Pi
(pH 8.0) and enzyme to give an absorbance change of 0.02/min. One unit
of enzyme activity is defined as the amount of enzyme that catalyzes
the conversion of 1 µmol of substrate to product/min. Protein
concentrations were determined as described by Bradford (16) with
bovine liver albumin as the standard.
-oxidation of 2,5,7-decatrienoyl-CoA was measured by incubating this
compound at concentrations between 5 and 50 µM in 0.2 M potassium Pi (pH 8.0) with 1 mM
NAD+, 0.3 mM CoASH, and a soluble extract of
rat liver mitochondria (4 µg/ml). The formation of NADH was
determined fluorometrically by excitation at 340 nm and by measuring
the emission at 460 nm with a PTI spectrofluorometer. The conversion of
2,5,7-decatrienoyl-CoA to 3,5,7-decatrienoyl-CoA was coupled to the
isomerization of the latter compound to the 2,4,6-isomer in the
presence of trienoyl-CoA isomerase (0.25 units/ml).
2,5,7-Decatrienoyl-CoA, at concentrations between 5 and 60 µM in 0.2 M potassium Pi (pH
8.0), was incubated with a soluble extract of rat liver mitochondria (4 µg/ml). The increase in absorbance at 337 nm was measured
spectrophotometrically. Rates were calculated based on an extinction
coefficient of 49,300 M
1 cm
1
(2).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation of
polyunsaturated fatty acids with conjugated double bonds at
odd-numbered positions is expected to produce 5,7-dienoyl-CoA
intermediates. To study the metabolism of these intermediates,
5,7-decadienoyl-CoA was synthesized. The required 5,7-decadienoic acid
was prepared from (4-carboxybutylidene)triphenylphosphorane and
2-trans-pentenal by a Wittig reaction (15). Since the
synthetic procedure is predicted to yield mostly the
trans-isomer of the newly formed double bond, the major
product is expected to be 5-trans,7-trans-decadienoic acid. The CoA
derivative of this acid was obtained in pure form after converting the
acid to the CoA thioester and isolating the major product by HPLC (Fig.
1A).
View larger version (24K):
[in a new window]
Fig. 1.
HPLC analysis of metabolites formed by
enzymatic conversions of 5,7-decadienoyl-CoA. A,
HPLC-purified 5,7-decadienoyl-CoA ( 5,7);
B, 3,5,7-decatrienoyl-CoA (
3,5,7)
formed from 5,7-decadienoyl-CoA (8 nmol in 0.2 ml of 0.1 M
potassium Pi (pH 8.0)) by acyl-CoA oxidase (0.1 unit) and
peroxisomal multifunctional protein I (6 µg) within 10 min;
C, 2,4,6-decatrienoyl-CoA (
2,4,6)
formed from 3,5,7-decatrienoyl-CoA, described for B, by
dienoyl-CoA isomerase (0.4 units); D, HPLC-purified
2,4,6-decatrienoyl-CoA after incubation without or with crotonase (0.7 units); E, 2,6-decadienoyl-CoA (
2,6)
formed from HPLC-purified 2,4,6-decatrienoyl-CoA (10 nmol in 0.5 ml of
0.1 M potassium Pi (pH 8.0)) by E. coli 2,4-dienoyl-CoA reductase (5 µg) plus 0.1 mM
NADPH within 5 min; F, 3-hydroxydec-6-enoyl-CoA
(3OH
6) formed from half of the sample described
for E by crotonase (0.7 units) within 1 min.
-oxidation of fatty acids that have
odd-numbered conjugated double bonds.
Rates of respiration supported by fatty acid oxidation in coupled
rat liver mitochondria
View larger version (28K):
[in a new window]
Fig. 2.
Spectrophotometric analysis of enzymatic
conversions of 5,7-decadienoyl-CoA and its metabolites.
A, spectral changes associated with the dehydrogenation of
5,7-decadienoyl-CoA (7 µM in 0.1 M potassium
Pi (pH 9)) by acyl-CoA oxidase (30 milliunits/ml) and
isomerization of 2,5,7-decatrienoyl-CoA to 3,5,7-decatrienoyl-CoA by
enoyl-CoA isomerase (0.15 µg/ml). Spectrum 1,
5,7-decadienoyl-CoA at time 0; spectrum 2, 30 s after
the addition of acyl-CoA oxidase; spectrum 3, 3 or 30 min
after the addition of acyl-CoA oxidase; spectrum 4, 30 s after the addition of enoyl-CoA isomerase to the sample characterized
by spectrum 3; spectrum 5, 2 min after the
addition of enoyl-CoA isomerase. B, spectral changes
associated with the conversion of 5,7-decadienoyl-CoA to
3,5,7-decatrienoyl-CoA catalyzed by acyl-CoA oxidase and enoyl-CoA
isomerase. Spectrum 1, 5,7-decadienoyl-CoA (19 µM in 0.1 M potassium Pi (pH
8.0)); spectrum 2, 15 s after the addition of acyl-CoA
oxidase (0.15 units/ml) and peroxisomal multifunctional protein I (10 µg/ml); spectra 3 and 4, 1 and 3 min after
enzyme addition, respectively. C, spectral changes
associated with the isomerization of 3,5,7-decatrienoyl-CoA to
2,4,6-decatrienoyl-CoA catalyzed by a soluble extract of rat liver
mitochondria or partially purified dienoyl-CoA isomerase.
Spectrum 1, 3,5,7-decatrienoyl-CoA (20 µM in
0.1 M potassium Pi (pH 8.0)); spectra
2-4, 3, 7, and 30 min after the addition of enzyme, respectively;
spectrum 5, HPLC-purified 2,4,6-decatrienoyl-CoA.
D, spectral changes associated with the reduction of
2,4,6-decatrienoyl-CoA by NADPH in the presence of purified
2,4-dienoyl-CoA reductase from E. coli. Spectrum
1, 2,4,6-decatrienoyl-CoA (4 µM in 0.1 M
potassium Pi (pH 8.0); NADPH (0.1 mM) was added
to the sample and reference solutions and the reaction was initiated by
the addition of 2,4-dienoyl-CoA reductase (0.5 µg)); spectra
2-5, 15 s, 1 min, 2.5 min, and 6 min after starting the
reaction, respectively.
-oxidation of 2,5,7-decatrienoyl-CoA in the presence of CoASH and
NAD+ were determined by measuring fluorometrically the
formation of NADH (Fig. 3, curve
A). The rates so obtained were compared with rates of
2,4,6-decatrienoyl-CoA formation determined spectrophotometrically at
340 nm in the absence of cofactors, but in the presence of excess
trienoyl-CoA isomerase (Fig. 3, curve B). Under these
conditions, the rate of the 2,5,7-trienoyl-CoA to 3,5,7-trienoyl-CoA
isomerization is measured. Since this isomerization is, for all
practical purposes, irreversible, it determines the fraction of
2,5,7-trienoyl-CoA that will be metabolized by way of the trienoyl-CoA
isomerase-dependent pathway. The direct
-oxidation was
found to be the favored pathway (Fig. 3). However, the degradation of
2,5,7-decatrienoyl-CoA via 2,4,6-decatrienoyl-CoA was found to make a
significant contribution to the total metabolism of
2,5,7-decatrienoyl-CoA, accounting for almost 50% of the total flux at
low and high substrate concentrations.
View larger version (17K):
[in a new window]
Fig. 3.
Rates of 2,5,7-decatrienoyl-CoA metabolism by
a soluble extract of rat liver mitochondria as a function of the
substrate concentration. Curve A, rate of NADH
formation in the presence of 1 mM NAD+ and 0.3 mM CoASH; curve B, rate of
2,4,6-decatrienoyl-CoA formation in the presence of trienoyl-CoA
isomerase (0.25 units/ml), but in the absence of coenzymes.
View larger version (19K):
[in a new window]
Fig. 4.
Immunoprecipitation of dienoyl-CoA and
trienoyl-CoA isomerase activities present in a partially purified
preparation of dienoyl-CoA isomerase by serum raised against purified
dienoyl-CoA isomerase from rat liver. For details, see
"Experimental Procedures." Shown are the activities of dienoyl-CoA
isomerase ( and
) and trienoyl-CoA isomerase (
and
)
remaining in the supernatant.
and
, antiserum;
and
,
preimmune serum.
Purification of dienoyl-CoA and trienoyl-CoA isomerases from pig heart
View larger version (25K):
[in a new window]
Fig. 5.
Analyses of fractions eluted from a reactive
red 120 column during the final purification step of trienoyl-CoA
isomerase. A, fractions were assayed for trienoyl-CoA
isomerase ( ) (values were multiplied by 75), dienoyl-CoA isomerase
(
), and protein (
) based on the relative densities of bands shown
in B. B, fractions were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE) and stained
for protein with Coomassie Brilliant Blue.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation. Chain shortening of such unsaturated fatty
acyl-CoAs by
-oxidation is expected to produce 5,7-dienoyl-CoA
intermediates (Fig. 6, compound II). Based on evidence
obtained with monounsaturated fatty acids that have a double bond at an
odd-numbered position (11), the degradation of 5,7-dienoyl-CoA is
predicted to proceed by two different routes. Common to both of them
would be the dehydrogenation of 5,7-dienoyl-CoA to 2,5,7-trienoyl-CoA
(Fig. 6, compound III) catalyzed by one of the acyl-CoA
dehydrogenases. If 2,5,7-trienoyl-CoA completes the cycle of
-oxidation, the resultant product would be 3,5-dienoyl-CoA. The
further metabolism of such intermediates has been shown to require the
sequential actions of dienoyl-CoA isomerase, 2,4-dienoyl-CoA reductase,
and enoyl-CoA isomerase to produce 2-trans-enoyl-CoA, which
can reenter the
-oxidation spiral (11). If, however, enoyl-CoA
isomerase catalyzes the isomerization of the double bond from the
2,3-position to the 3,4-position, 3,5,7-trienoyl-CoA (Fig. 6,
compound IV) would be formed. This isomerization would be
irreversible for all practical purposes, as is the isomerization of
2,5-octadienoyl-CoA to 3,5-octadienoyl-CoA (13). The evidence presented
in this report indicates that a significant fraction of the
2,5,7-trienoyl-CoA is converted to the 3,5,7-isomer even though the
major portion of 2,5,7-trienoyl-CoA completes the cycle of
-oxidation and thereby bypasses the formation of a trienoyl-CoA intermediate. The further metabolism of 3,5,7-trienoyl-CoA was hitherto
unknown. The identification of trienoyl-CoA isomerase suggested a
pathway for the complete degradation of 3,5,7-trienoyl-CoAs. Isomerization of 3,5,7-trienoyl-CoA by trienoyl-CoA isomerase yields
2,4,6-trienoyl-CoA, presumably in the
all-trans-configuration, as established for the formation of
2,4-octadienoyl-CoA from 3,5-octadienoyl-CoA (Fig. 6, compound
V). In liver mitochondria, 2,4,6-trienoyl-CoA can be reduced to
3,6-dienoyl-CoA by NADPH-dependent 2,4-dienoyl-CoA reductase (2). The complete degradation of the latter intermediate would proceed by well established reactions that require the actions of
enoyl-CoA isomerase to shift the odd-numbered double bond from carbon 3 to 2 and of 2,4-dienoyl-CoA reductase to reductively remove the
even-numbered double bond.
View larger version (17K):
[in a new window]
Fig. 6.
Proposed trienoyl-CoA
isomerase-dependent pathway for the
-oxidation of
9-cis,11-trans-octadecadienoyl-CoA
(conjugated linoleoyl-CoA).
The co-purification of trienoyl-CoA and dienoyl-CoA isomerases as well as the co-immunoprecipitation of these two enzyme activities by antibodies raised against dienoyl-CoA isomerase strongly suggested that both enzymes reside on one protein. The demonstration that one protein, as indicated by a single band on SDS-polyacrylamide gel electrophoresis, exhibited both trienoyl-CoA and dienoyl-CoA isomerase activities confirmed the conclusion about the association of both enzyme activities with one protein.
Dienoyl-CoA isomerase was first isolated from rat liver mitochondria and reported to have a subunit molecular mass of 32 kDa (10). Subsequently, peroxisomes were shown to contain a form of this enzyme that cross-reacted with antibodies raised against the mitochondrial enzyme (19). The molecular cloning of dienoyl-CoA isomerase yielded a cDNA sequence that strongly suggested peroxisomal as well as mitochondrial localizations of this enzyme (20). Evidence in support of the dual subcellular localization of this protein was obtained by immunoelectron microscopy. However, the precise terminal sequences of the mature forms of dienoyl-CoA isomerase detected in mitochondria and peroxisomes have not been reported.
Since the published cDNA sequence encodes a 36-kDa polypeptide,
molecular masses >36 kDa reported for this enzyme (19, 21) must be
those of other polypeptides. Finally, expression of a fragment of the
cDNA coding for dienoyl-CoA isomerase yielded an active protein
that was crystallized and analyzed by x-ray diffraction (22). The
crystal structure revealed an active-site pocket that is hydrophobic
except for the side chains of three acidic residues. Two of these
residues, Glu-196 and Asp-204, were proposed to facilitate the proton
removal from carbon 2 and the proton addition to carbon 6, respectively, of the substrate, 3,5-dienoyl-CoA. If 3,5,7-trienoyl-CoA
binds to the same active site, the proton abstraction from carbon 2 could also be facilitated by Glu-196. However, the residue involved in
the protonation of carbon 8 remains to be identified.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant HL 30847 from NHLBI, National Institutes of Health; by United States Public Health Service Grant RR 03060 to Research Centers of Minority Institutions; and by a grant from the City University of New York PSC-CUNY Research Award Program.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, City University of New York, Convent Ave. at 138th St.,
New York, NY 10031. Tel.: 212-650-8323; Fax: 212-650-8322.
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: HPLC, high performance liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|