INTRODUCTION

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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

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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.

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 KP_i (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 KP_i, (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 M¹ cm¹ 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 KP_i, (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 M¹ cm¹ 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 KP_i (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 M¹ cm¹ 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 M¹ cm¹ 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 M¹ cm¹ was used to calculate rates. A standard assay contained 0.2 M KP_i (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.

Metabolic Studies-- For rate measurements, various amounts of 2,5-octadienoyl-CoA in 0.2 M KP_i (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 M¹ cm¹ 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 M¹ cm¹ 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 KP_i (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 M¹ cm¹ 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 KP_i (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 KP_i (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

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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 K_m and V_max 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 V_max was observed when the substrate contained the 5-cis double bond, while the K_m was virtually unaffected. The opposite observation was made with 3-hydroxyacyl-CoA dehydrogenase, which yielded a 4-fold higher K_m 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 K_m 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 V_max and K_m) 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 (3OH5); 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 ^3,52,5 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

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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 V_max value determined with 3-keto-5-octenoyl-CoA as the substrate was almost 6 times lower than the V_max obtained with 3-ketooctanoyl-CoA. In addition, the K_m for the substrate was 5 times higher when a 5-cis double bond was present in the acyl chain. In contrast, the V_max of 3-hydroxyacyl-CoA dehydrogenase was unaffected by the presence of a 5-cis double bond, whereas the K_m 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 ^2,53,5 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.