Metabolism of 4-pentenoic acid and inhibition of thiolase by metabolites of 4-pentenoic acid

The metabolism of 4-pentenoic acid, a hypoglycemic agent and inhibitor of fatty acid oxidation, has been studied in rat heart mitochondria. Confirmed was the conversion of 4-pentenoic acid to 2,4-pentadienoyl coenzyme A (CoA), which either is directly degraded via beta-oxidation or is first reduced in a NADPH-dependent reaction before it is further degraded by beta-oxidation. At pH 6.9, the NADPH-dependent reduction of 2,4-pentadienoyl-CoA proceeds 10 times faster than its degradation by beta-oxidation. At pH 7.8, this ratio is only 2 to 1. The direct beta-oxidation of 2,4-pentadienoyl-CoA leads to the formation of 3-keto-4-pentenoyl-CoA, which is highly reactive and spontaneously converts to another 3-ketoacyl-CoA derivative (compound X). 3-Keto-4-pentenoyl-CoA is a poor substrate of 3-ketoacyl-CoA thiolase (EC 2.3..1.16) whereas compound X is not measurably acted upon by this enzyme. The effects of several metabolites of 4-pentenoic acid on the activity of 3-ketoacyl-CoA thiolase were studied. 3,4-Pentadienoyl-CoA is a weak inhibitor of this enzyme that is protected against the inhibition by acetoacetyl-CoA. The most effective inhibitor of 3-ketoacyl-CoA thiolase was found to be 3-keto-4-pentenoyl-CoA, which inhibits the enzyme in both a reversible and irreversible manner. The reversible inhibition is possibly a consequence of the inhibitor being a poor substrate of 3-ketoacyl-CoA thiolase. It is concluded that 4-pentenoic acid is metabolized in mitochondria by two pathways. The minor yields 3-keto-4-pentenoyl-CoA, which acts both as a reversible and as a irreversible inhibitor of 3-ketoacyl-CoA thiolase and consequently of fatty acid oxidation.     

Inhibition of brain glutamate decarboxylase by 2-keto-4-pentenoic acid, a metabolite of allylglycine.

Abstract— 2-Keto-4-pentenoic acid, a potent inhibitor of brain glutamate decarboxylase (Orlowski et al., 1977) was prepared by oxidative deamination of l -allylglycine with snake venom l -amino acid oxidase. In the presence of glutamate the keto acid is a competitive inhibitor of the enzyme with respect to glutamate; its K_i is 2.4 ± 10^?6m . After preincubation of brain glutamate decarboxylase with 2-keto-4-pentenoic acid in the absence of glutamate, a slow and incomplete reactivation is obtained by prolonged dialysis, Sephadex gel-filtration, and dilution, suggesting the formation of a slowly dissociating enzyme-inhibitor complex and partial inactivation of the enzyme. In vivo inhibition of brain glutamate decarboxylase after administration of allylglycine is maximal after 2-8 h with activity returning to normal after 16 h. The inhibition of the enzyme after administration of d -allylglycine was greatest in the cerebellum and the medulla-pons area, the sites of the highest activity of d -amino acid oxidase. These results are interpreted as strongly supporting the postulate that allylglycine-induced inhibition of brain glutamate decarboxylase is due to the in vivo formation of 2-keto-4-pentenoic acid.     

Mitochondrial metabolism of valproic acid

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

REGIONAL CHANGES IN CEREBRAL GABA CONCENTRATION AND CONVULSIONS PRODUCED BY d AND BY l-ALLYLGLYCINE

Abstract— The convulsant action of allylglycine (2-amino-4-pentenoic acid) is due to the metabolic conversion of allylglycine to 2-keto-4-pentenoic acid, a more potent glutamic acid decarboxylase inhibitor and more potent convulsant than the parent compound. We report regional changes in cerebral GABA concentration in rats after administration of d - and l -allylglycine. d -Allylglycine (3.75 mmol/kg) induced convulsions in 95–115 min, characterised by repeated clonic limb movements and rapid rotation around the head to tail axis. GABA concentrations were only reduced in cerebellum and ponsmedulla during the pre and post-convulsive periods. The localised reduction of GABA concentration is consistent with the enzymic conversion of d -allylglycine to 2-keto-4-pentenoic acid catalysed by cerebral d -amino acid oxidase, an enzyme known to be localised to the hind brain and spinal cord. l -allylglycine (1.2mmol/kg i.p.) induced convulsions in 65 -90 min, characterised by violent running followed by tonic flexion and extension. During the pre-convulsive period, GABA concentrations were reduced in all brain areas studied except the globus pallidus and ventral midbrain. The widespread decreases in GABA concentration suggest that the enzyme(s) which catalyse the conversion of l -allylglycine to 2-keto-4-pentenoic acid are widely distributed within the brain.     

Conditions for the self-catalysed inactivation of carnitine acetyltransferase. A novel form of enzyme inhibition

1. Carnitine acetyltransferase is very rapidly inhibited in the presence of bromoacetyl-(-)-carnitine plus CoA or of bromoacetyl-CoA plus (-)-carnitine. 2. Under appropriate conditions, the enzyme may be titrated with either bromoacetyl substrate analogue; in each case about 1mole of inhibitor is required to inactivate completely 1mole of enzyme of molecular weight 58000+/-3000. 3. Inhibition by bromoacetyl-CoA plus (-)-carnitine results in the formation of an inactive enzyme species, containing stoicheiometric amounts of bound adenine nucleotide and (-)-carnitine in a form that is not removed by gel filtration. This is shown to be S-carboxymethyl-CoA (-)-carnitine ester. 4. The inhibited enzyme recovers activity slowly on prolonged standing at 4 degrees . 5. Incubation with S-carboxymethyl-CoA (-)-carnitine ester causes a slow inhibition of carnitine acetyltransferase. 6. The formation of bound S-carboxymethyl-CoA (-)-carnitine ester by the enzyme is discussed. Presumably the resulting inhibition reflects binding of the ester to both the CoA- and carnitine-binding sites on the enzyme and its consequent very slow dissociation. These observations confirm that carnitine acetyltransferase can form ternary enzyme-substrate complexes; this also appears to be the case with carnitine palmitoyltransferase and choline acetyltransferase.     

Dehalogenation and Deamination of l-2-Amino-4-chloro-4-pentenoic Acid by Proteus mirabilis

Eighty-one strains of bacteria were tested for their ability to catalyze the release of chloride ion from Dl-2-amino-4-chloro-4-pentenoic acid. A dehalogenating enzyme was obtained from the cells of Proteus mirabilis IFO 3849, which can use the l-isomer. The enzyme was constitutively produced. The conversion of l-2-amino-4-chloro-4-pentenoic acid to 2-keto-4-pentenoic acid, ammonia, and chloride ion was demonstrated. The reaction product, 2-keto-4-pentenoic acid, was isolated as its 2,4-dinitrophenylhydrazone and identified by catalytic hydrogenolysis of the hydrazone to the corresponding amino acid, norvaline.     

4-Bromo-2-octenoic acid specifically inactivates 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation in rat liver mitochondria

In an attempt to develop a compound which would specifically inhibit 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in whole mitochondria, 4-bromo-2-octenoic acid was synthesized and studied. After rat liver mitochondria were preincubated with 4-bromo-2-octenoic acid for 3 min, respiration supported by either palmitoylcarnitine or pyruvate was completely abolished, whereas no inhibition was observed with rat heart mitochondria. Addition of carnitine stimulated respiration supported by pyruvate without relieving inhibition of palmitoylcarnitine-dependent respiration. Hence, this compound seems to be a specific inhibitor of beta-oxidation. When the enzymes of beta-oxidation were assayed in a soluble extract prepared from mitochondria preincubated with 4-bromo-2-octenoic acid, only 3-ketoacyl-CoA thiolase was found to be inactivated. 4-Bromo-2-octenoic acid is metabolized by mitochondrial beta-oxidation enzymes to 3-keto-4-bromooctanoyl-CoA which effectively and irreversibly inhibits 3-ketoacyl-CoA thiolase but not acetoacetyl-CoA thiolase (EC 2.3.1.9). Even though 3-keto-4-bromooctanoyl-CoA inhibits the latter enzyme reversibly, 4-bromo-2-octenoic acid does not inhibit ketogenesis in rat liver mitochondria with acetylcarnitine as a substrate. It is concluded that 4-bromo-2-octenoic acid specifically inhibits mitochondrial fatty acid oxidation by inactivating 3-ketoacyl-CoA thiolase in rat liver mitochondria.     

Specific alkylation of a histidine residue in carnitine acetyltransferase by bromoacetyl-l-carnitine

Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[(14)C]bromoacetyl-l-carnitine yields N-3-carboxy[(14)C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue.     

Sequence of reactions which follows enzymatic oxidation of allylglycine.

The pathway following flavoprotein-catalyzed oxidation of allylglycine (2-amino-4-pentenoate) has been studied and found to be dependent on the incubation conditions. In N-2-hydroxyethyl-N'-2-ethanesulfonic acid (Hepes) buffer, the oxidation product 2-iminium-4-pentenoate predominantly reacts to form 2-amino-2,4-pentadienoate, a strong noncovalent inhibitor of D-amino-acid oxidase. However, in pyrophosphate buffer, the more rapid reaction is hydrolysis to form 2-keto-4-pentenoate, which has been found to be a substrate for L-lactic dehydrogenase. 2-Keto-4-pentenoate is in rapid equilibrium with 2-hydroxy-2,4-pentadienoate, which is also a strong noncovalent inhibitor of D-amino-acid oxidase. In both systems, these metastable intermediates react in subsequent slower steps to yield trans-2-keto-3-pentenoate, which accumulates in the incubation. Syntheses of trans-2-amino- and trans-2-keto-3-pentenoate are described. Comparisons between the reactivities of acetylenic and olefinic species have been made based on the differences between this pathway and that following oxidation of propargylglycine [Marcotte, P., and Walsh, C. (1978), Biochemistry 17 (preceding paper in this issue)].     

Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine.

A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide.     

Inhibitors of fatty acid oxidation

This review discusses inhibitors of fatty acid oxidation for which sites and mechanisms of inhibition are reasonably well understood. Included in this review are hypoglycin, an inhibitor of butyryl-CoA dehydrogenase (EC 1.3.99.2), 4-pentenoic acid, 2-bromooctanoic acid, and 4-bromocrotonic acid all of which inhibit mitochondrial thiolases (EC 2.3.1.9 and 2.3.1.16) as well as several inhibitors of carnitine palmitoyltransferase I (EC 2.3.1.21) as for example 2-tetradecylglycidic acid, 2-bromopalmitic acid and aminocarnitine. Most of these inhibitors of fatty acid oxidation have been shown to cause hypoglycemia in animals and some also cause hypoketonemia. The advantages and limitations of using these inhibitors in metabolic studies are discussed.     

Accumulation of polyhydroxyalkanoic acid containing large amounts of unsaturated monomers in Pseudomonas fluorescens BM07 utilizing saccharides and its inhibition by 2-bromooctanoic acid.

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C(7)) to hexadecanoic acid (C(16)) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C(18)) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit beta-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 microM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant K(i) (the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 microM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin.     

DL-aminocarnitine and acetyl-DL-aminocarnitine. Potent inhibitors of carnitine acyltransferases and hepatic triglyceride catabolism

DL-Aminocarnitine (3-amino-4-trimethylaminobutyric acid) and acetyl-DL-aminocarnitine (3-acetamido-4-trimethylaminobutyric acid) have been synthesized and the interactions of these compounds with carnitine acetyltransferase and carnitine palmitoyltransferase investigated. As anticipated from the low group transfer potential of amides, carnitine acetyltransferase catalyzes the transfer of acetyl groups from CoASAc to aminocarnitine (Km = 3.8 mM) but does not catalyze detectable transfer from acetylaminocarnitine to CoASH. Acetyl-DL-aminocarnitine is, however, a potent competitive inhibitor of carnitine acetyltransferase (Ki = 24 microM) and is bound to carnitine acetyltransferase about 13-fold more tightly than is acetylcarnitine, with which it is isosteric. DL-Aminocarnitine and, to a lesser extent, acetyl-DL-aminocarnitine are also inhibitors of the carnitine palmitoyltransferase activity of detergent-lysed rat liver mitochondria; in the presence of 1 mM L-carnitine, 5 microM aminocarnitine inhibits palmitoyl transfer by 64%. Significant acylation of aminocarnitine by palmitoyl-CoA was not observed. Neither aminocarnitine nor acetylaminocarnitine is significantly catabolized by mice; aminocarnitine is converted to acetylaminocarnitine in vivo. Both compounds are excreted in the urine. Mice given acetylaminocarnitine catabolize [14C]acetyl-L-carnitine and [14C]palmitate to 14CO2 more slowly than do control animals. Mice given acetylaminocarnitine and then starved are found to reversibly accumulate triglycerides in their livers; mice given the inhibitor but not starved do not show this effect.     

Inhibition of carnitine acetyltransferase by bile acids: implications for carnitine analysis

Carnitine acetyltransferase is used in a radioenzymatic assay to measure the concentration of carnitine. While determining the concentration of carnitine in rat bile, we found that the apparent concentration increased as bile was diluted (6.7 +/- 1.0 and 66.6 +/- 9.4 nmol/ml in undiluted and 20-fold diluted bile, respectively). The present study was designed to investigate whether a component of bile inhibited carnitine acetyltransferase. Inhibition was evaluated by measuring carnitine concentration in bile or by determining the recovery of a known amount of carnitine in the presence of bile. Inhibitory activity was extractable in organic solvents, stable to heat and base treatments, resistant to trypsin and lipase digestions, and removable by cholestyramine, a bile acid-binding resin. These results suggested that the inhibitory activity was associated with bile acids. Direct evidence was obtained by showing a reduced detectability of carnitine in the presence of individual bile acids. Chenodeoxycholic acid was the most potent inhibitor. Inhibition was unrelated to the detergent properties of bile acids. Kinetic studies revealed that carnitine acetyltransferase was inhibited competitively by chenodeoxycholic acid with a Ki of 520 microM. Bile acids also interfered in the quantitation of carnitine in cholestatic plasma. Carnitine concentration in such plasma was underestimated (17.5 +/- 2.1 mmol/ml). Reduction of bile acid concentration by a 20-fold dilution of cholestatic plasma resulted in a 3-fold higher carnitine concentration (54.6 +/- 9.0 nmol/ml). Results demonstrate that, because of the inhibition of carnitine acetyltransferase by bile acids, the radioenzymatic assay will underestimate carnitine concentration in bile or in cholestatic plasma. Accurate measurement requires either the removal of bile acids or a marked reduction in their concentration.     

The effects of substrates on the optical rotatory dispersion of carnitine acetyltransferase

1. The optical rotatory dispersion of carnitine acetyltransferase is altered in the presence of l-carnitine or acetyl-l-carnitine. These changes, which include an increase in the reduced mean residue rotation at 233nm. ([M'](233)), suggest that substrate binding causes the enzyme to unfold. 2. CoA and acetyl-CoA have no immediate effect on [M'](233) and CoA has no effect on the change in this parameter induced by l-carnitine. 3. The change in [M'](233) was used as a measure of the degree of saturation of the enzyme with carnitine substrates. Dissociation constants for the enzyme complexes with l-carnitine, d-carnitine and acetyl-l-carnitine were determined in this way. 4. Prolonged incubation of carnitine acetyltransferase in the presence of CoA leads to a small increase in the value of [M'](233) accompanied by irreversible inhibition of the enzyme. 5. Optical-rotatory-dispersion studies of two specifically inhibited enzyme forms are reported.     

Two mechanisms produce tissue-specific inhibition of fatty acid oxidation by oxfenicine.

Oxfenicine [S-2-(4-hydroxyphenyl)glycine] is transaminated in heart and liver to 4-hydroxyphenylglyoxylate, an inhibitor of fatty acid oxidation shown in this study to act at the level of carnitine palmitoyltransferase I (EC 2.3.1.21). Oxfenicine was an effective inhibitor of fatty acid oxidation in heart, but not in liver. Tissue specificity of oxfenicine inhibition of fatty acid oxidation was due to greater oxfenicine transaminase activity in heart and to greater sensitivity of heart carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate [I50 (concentration giving 50% inhibition) of 11 and 510 microM for the enzymes of heart and liver mitochondria, respectively]. Branched-chain-amino-acid aminotransferase (isoenzyme I, EC 2.6.1.42) was responsible for the transamination of oxfenicine in heart. A positive correlation was found between the capacity of various tissues to transaminate oxfenicine and the known content of branched-chain-amino-acid aminotransferase in these tissues. Out of three observed liver oxfenicine aminotransferase activities, one may correspond to asparagine aminotransferase, but the major activity could not be identified by partial purification and characterization. As reported previously for malonyl-CoA inhibition of carnitine palmitoyltransferase I, 4-hydroxyphenylglyoxylate inhibition of this enzyme was found to be very pH-dependent. In striking contrast with the kinetics of malonyl-CoA inhibition, 4-hydroxyphenylglyoxylate inhibition was not affected by oleoyl-CoA concentration, but was partially reversed by increasing carnitine concentrations.     

The substrate specificity of carnitine acetyltransferase

1. A study of the acyl group specificity of the carnitine acetyltransferase reaction [acyl-(-)carnitine+CoASH right harpoon over left harpoon (-)-carnitine+acyl-CoA] has been made with the enzyme from pigeon breast muscle. Acyl groups containing up to 10 carbon atoms are transferred and detailed kinetic investigations with a range of acyl-CoA and acylcarnitine substrates are reported. 2. Acyl-CoA derivatives with 12 or more carbon atoms in the acyl group are potent reversible inhibitors of carnitine acetyltransferase, competing with acetyl-CoA. Lauroyl- and myristoyl-CoA show a mixed inhibition with respect to (-)-carnitine, but palmitoyl-CoA competes strictly with this substrate also. Palmitoyl-dl-carnitine shows none of these effects. 3. Ammonium palmitate inhibits the enzyme competitively with respect to (-)-carnitine and non-competitively with respect to acetyl-CoA. 4. It is suggested that a hydrophobic site exists on the carnitine acetyltransferase molecule. The hydrocarbon chain of an acyl-CoA derivative containing eight or more carbon atoms in the acyl group may interact with this, which results in enhanced acyl-CoA binding. Competition occurs between ligands bound to this hydrophobic site and the carnitine binding site. 5. The possible physiological significance of long-chain acyl-CoA inhibition of this enzyme is discussed.     

Accumulation of Polyhydroxyalkanoic Acid Containing Large Amounts of Unsaturated Monomers in Pseudomonas fluorescens BM07 Utilizing Saccharides and Its Inhibition by 2-Bromooctanoic Acid

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C₇) to hexadecanoic acid (C₁₆) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C₁₈) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit β-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 μM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant K_i (the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 μM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin.     

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of M_r 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.