butyryl-CoA Synthetase Research Articles

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.

Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome

Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. "Activation" of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways. The availability of the sequenced human genome has facilitated the investigation of the number of ACS genes present. Using two conserved amino acid sequence motifs to probe human DNA databases, 26 ACS family genes/proteins were identified. ACS activity in either humans or rodents was demonstrated previously for 20 proteins, but 6 remain candidate ACSs. For two candidates, cDNA was cloned, protein was expressed in COS-1 cells, and ACS activity was detected. Amino acid sequence similarities were used to assign enzymes into subfamilies, and subfamily assignments were consistent with acyl chain length preference. Four of the 26 proteins did not fit into a subfamily, and bootstrap analysis of phylograms was consistent with evolutionary divergence. Three additional conserved amino acid sequence motifs were identified that likely have functional or structural roles. The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate. Knowing the full complement of ACS genes in the human genome will facilitate future studies to characterize their specific biological functions.

Mucosal enzyme activity for butyrate oxidation; no defect in patients with ulcerative colitis.

Butyrate is an important energy source for the colon and its metabolism has been reported to be defective in ulcerative colitis. One mechanism for defective butyrate metabolism in patients with ulcerative colitis could be an enzyme deficiency in the beta-oxidation pathway of butyrate. This study was undertaken to measure the activity of each enzyme involved in the beta-oxidation pathway of butyrate in colonic epithelium. Patients with ulcerative colitis (n = 33), Crohn's colitis (n = 10), and control subjects with colorectal cancer or diverticular disease (n = 73) were studied. Analysis was carried out using fluorometric and spectrophotometric techniques on homogenised epithelial biopsy specimens. Significantly increased butyryl CoA dehydrogenase activity was found in mucosa from patients with ulcerative colitis (33.2 (28.3, 38.1) mumol/g wet weight/min:mean (95% CI)) compared with activity in mucosa from control patients (24.3 (20.9, 27.7) mumol/g wet weight/min:mean (95% CI)) p < 0.02. No significant increase in activity of the enzymes butyryl-CoA synthetase, crotonase or hydroxybutyryl-CoA dehydrogenase was found in patients with ulcerative colitis. In contrast the mucosal thiolase activity was significantly lower in those patients with quiescent colitis (3.21 (2.61, 3.81) mumol/g wet weight/min:mean (95% CI)) when compared with control mucosa (5.69 (5.09, 6.29) mumol/g wet weight/min:mean (95% CI)) p < 0.001. However, mucosal thiolase activity increases with the age of the donor patient and differences in the age range of the patient groups probably account for this finding. This study shows no substantial deficiency of enzyme activity in the beta-oxidation pathway of butyrate in the mucosa of patients with ulcerative colitis in histological remission.

Substrate and hormone regulation of palmitoyl-CoA synthetase in 7800 C1 Morris hepatoma cells and cultured rat hepatocytes

The effects of tetradecylthioacetic acid (TTA), insulin and dexamethasone on palmitoyl-CoA synthetase activity and its mRNA both in 7800 Cl hepatoma cells and cultured rat hepatocytes were studied. (1) When the hepatoma cells were cultivated in the presence of fatty acids or alkyl thioacetic acids (3-thia fatty acids) palmitoyl-CoA synthetase activity was increased several fold. The stronger effect was obtained with TTA, which also increased long-chain acyl-CoA synthetase mRNA significantly. TTA has no inducing effect on butyryl-CoA synthetase and little effect on octanoyl-CoA synthetase in the same cells. Dexamethasone also had inducing effect on palmitoyl-CoA synthetase in the hepatoma cells. Insulin counteracted the induction given by TTA. All of these regulation actions take place at the pretranslational level. (2) In isolated hepatocytes the activity of palmitoyl-CoA synthetase was much higher than in hepatoma cells, but it was lost rapidly in culture. The loss of the enzyme activity was slowed down in the presence of TTA and insulin, either alone or combined. Dexamethasone combined with TTA reversed the loss of enzyme activity, while dexamethasone alone even increased the loss. Analysis of palmitoyl-CoA synthetase mRNA shows that TTA prevents the loss of the enzyme activity by inducing mRNA of the enzyme, dexamethasone enhances the effect of TTA, while insulin stabilizes the enzyme activity in the cultured cells without increasing the mRNA level.

Influence of dietary forage and energy intake on metabolism and acyl-CoA synthetase activity in bovine ruminal epithelial tissue

Twenty calves (7 mo old) were blocked by breed, sex, and weight into five groups of four calves and randomly assigned to either a 90% forage (alfalfa) or a 90% grain (50% sorghum and 50% wheat) diet fed at one (1M) or two (2M) times NEm for 140 d. Samples of ruminal epithelial tissue were collected from the anterior ventral sac, and papillae were cut free by hand and used for in vitro incubations and acyl-CoA synthetase assays. Substrates were acetate (90 mM), propionate (60 mM), butyrate (30 mM), and glucose (20 mM). Net productions of beta-hydroxybutyrate and acetoacetate from acetate were greater (P less than .01) with the 2M feeding; however, 14CO2 production from acetate was greater (P less than .05) with the grain diet. Net production of lactate (P = .09) and pyruvate (P less than .01) from propionate increased with the 2M feeding, whereas net lactate production from glucose decreased (P less than .01). Uptakes of VFA were similar with 1M and 2M feeding and were about 10-fold greater than uptakes of glucose. Production of 14CO2 from propionate was two- to fivefold greater than from acetate, butyrate, or glucose. Oxygen consumption was greater (P less than .01) with 2M feeding and unaffected by substrate. Activities of butyryl-CoA synthetase (nmol.mg of tissue-1.h-1) were greater (P less than .05) for animals consuming the forage diets. Addition of butyrate inhibited acetyl- and propionyl-CoA synthetase activity by 63 and 92%, respectively for all dietary treatments. Overall, metabolism of ruminal epithelial tissue tended to increase with the 2M feeding. Influence of dietary forage content on metabolism and activity of acyl-CoA synthetases was minimal, but the high-forage diet caused slight increases.

Purification and kinetic properties of butyryl-CoA synthetase from Paecilomyces varioti.

A butyryl-CoA synthetase (EC 6.2.1.2) was obtained from Paecilomyces varioti AHU 9417 in a highly purified form, which appeared to be homogeneous on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate (SDS). The purified enzyme exhibited molecular weights of 145,000 for the native enzyme and 74,000 for the subunit. This enzyme required both monovalent and divalent cations for the reaction. The enzyme was active toward saturated fatty acids with 3 to 5 carbon atoms, and butyrate was the best substrate. The reaction mechanism of the enzyme was also investigated and was found to conform to a Bi-Uni-Uni-Bi ping-pong mechanism, in which the order of addition and evolution of substrates and products was ATP, butyrate, PPi, CoA, butyryl-CoA (or AMP) and AMP (or butyryl-CoA). Butyryl-CoA and AMP were evolved at random or at the same time.

The role of the available sulphydryl group in liver butyryl-coenzyme a synthetase

1. 1. Butyryl-CoA synthetase from acetone-dried ox liver mitochondria can be activated up to 80% by low concentrations of iodoacetate with the concomitant modification of one available enzyme thiol group. 2. 2. The relative molecular mass of the carboxymethylated enzyme was unchanged, and there were no major changes in the complex kinetic properties of the enzyme. 3. 3. Extraction experiments on liver mitochondria indicate that in vivo the enzyme is linked to a mitochondrial membrane by a bond cleavable by disulphide bond reducing agents.

Ox liver butyryl-coenzyme a synthetase—a subunit enzyme?

1. 1. Butyryl-CoA synthetase extracted from untreated or freeze-dried liver mitochondria consisted of two fractions of M, 40,000 and 46,000 as determined by gel permeation chromatography, the higher value Being connrmed Dy sedimentation equilibrium. 2. 2. A red pigment having the characteristics of haem was associated with the enzyme. This appeared to be an artifact of isolation. 3. 3. A number of active bands were obtained on polyacrylamide gel electrophoresis and isoelectric focusing, and on treatment with sodium dodecylsulphate or 6 M guanidine hydrochloride both dissociation and aggregation of the enzyme fractions occurred. 4. 4. As no evidence of protease contamination or proteolytic action could be detected, it is suggested that the active enzymes contain more than one polypeptide chain.

Regulation of Volatile Fatty Acid Uptake by Mitochondrial Acyl CoA Synthetases of Bovine Liver1,2

Mitochondria of bovine liver contain acyl CoA synthetases necessary for the uptake of propionate, butyrate, and valerate whereas acetate is bound only weakly. Purification of these enzymes separated a distinct propionyl CoA synthetase highly specific for propionate and acrylate and a butyrate-activating fraction with broad substrate specificity for short and medium chain fatty acids. Evidence from kinetic studies and sucrose density centrifugation suggested that this latter fraction was composed of two enzymes, a butyryl CoA synthetase and a valeryl CoA synthetase. The apparent molecular weights of the propionyl, butyryl, and valeryl CoA synthetases were 72,000, 67,000, and 65,000. The Michaelis-Menten constants of propionyl CoA synthetase for propionate, adenosine 5’-triphosphate, and coenzyme A were 1.3×10−3M, 1.3×10−3M, and 6.3×10−4M. Enzyme activity is regulated by the concentration of propionate in portal blood. Relative to propionyl, butyryl, or valeryl CoA synthetases little acetyl CoA synthetase could be demonstrated.In ruminants hepatic metabolism is such that use of acetate as an energy source is minimum. This ensures that an alternative energy source to glucose, as acetate units, will reach the extrahepatic tissues. Separation of a distinct propionyl CoA synthetase regulated by the concentration of propionate in portal blood is significant because a primary role of ruminant liver is to synthesize glucose from ruminally derived propionate.

Regulation of Volatile Fatty Acid Uptake by Mitochondrial Acyl CoA Synthetases of Bovine Heart1,2

Purification of components of heart mitochondria activating short chain fatty acids prepared from tissue of lactating Holstein cows demonstrated predominantly one acyl CoA synthetase, acetyl CoA synthetase activating acetate, and propionate. Activity of butyryl CoA synthetase was low. Propionyl CoA synthetase characteristically in bovine liver and kidney tissue could not be demonstrated in heart mitochondria. Thus, of the ruminally derived volatile fatty acids only acetate can be used by heart mitochondria as a primary energy source because of small quantities of propionate in peripheral blood.Acetyl CoA synthetase was a glycoprotein composed of a single polypeptide chain of apparent molecular weight 67,500. The Michaelis-Menten constant for acetate was 1.8×10−4M. By comparison with literature for blood acetate concentration we concluded that enzyme is saturated with substrate at all physiological concentrations of acetate. These kinetic properties ensure a constant supply of acetate as an energy source for maintaining heart function in ruminants.

Butyryl-CoA synthetase of pseudomonas aeruginosa —Purification and characterization

The preparation of highly purified medium chain acyl-CoA synthetase (Acid: CoA ligase, AMP-forming (EC 6.2.1.2)) from the cell extracts of Pseudomonas aeruginosa is described. The enzyme is inducibly formed in the cells of the microorganism, when it is grown with butyrate as a major carbon source. The purified enzyme is homogeneous on disc gel electrophoresis. Its molecular weight is approximately 142,000, and it is possibly composed of 4 identical subunits of approximately 37,000 molecular weight and has isoelectric point of 4.3. The enzyme catalyzes the stoichiometric conversion of butyrate and CoA to butyryl-CoA in the presence of ATP and Mg 2+. It also activates fatty acids with carbon chain lengths of 3 to 5 well, but is inactive toward fatty acids with carbon chain lengths of more than 6. The enzyme is sulfhydryl dependent and inactivated by silver and mercury compounds.

50] 4-Pentenoylcarnitine, cyclopropanecarbonylcarnitine, and cyclobutanecarbonylcarnitine

Publisher Summary This chapter discusses the preparation and effects of 4-pentenoylcarnitine, cyclopropanecarbonylcarnitine, and cyclobutanecarbonylcarnitine. Carnitine esters (carbonyl O -acylcarnitines) are synthesized and purified by reaction of the acid chloride of the acids with carnitine. 4-Pentenoic acid, cyclopropanecarboxylic acid, cyclopropanecarbonyl chloride, cyclo-butanecarboxylic acid, and cyclobutanecarbonyl chloride are available commercially. The ester bond content of preparations of carnitine esters can be determined by the hydroxamate method, which usually gives more than 97% of the theoretical value. Unreacted L -carnitine can be determined by reaction with acetyl-CoA, catalyzed by carnitine acetyltransferase, in the direction of acetylcarnitine synthesis by measuring the CoASH released using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). 4-Pentenoic acid, cyclopropanecarboxylic acid, and cyclobutanecarboxylic acid have little effect on mitochondrial reactions unless they are converted to their CoA esters in the matrix by the action of butyryl-CoA synthetase, which catalyzes a reaction that also requires ATP. L -Cyclopropanecarbonylcarnitine has been identified as a metabolite of the miticide cycloprate in animal tissues, milk, and urine, and in the eggs of phytophagousmites.

Mechanisms of the metabolic disturbances caused by hypoglycin and by pent-4-enoic acid in vitro studies

1. In the presence of the hypoglycin metabolites methylenecyclopropylpyruvate (MCPP) and methylenecyclopropylacetate (MCPA), rat liver mitochondria oxidized palmitoyl-carnitine only as far as butyrate and at a decreased rate. Although pent-4-enoic acid (pent-4-enoate) inhibited the rate of β-oxidation of palmitoyl-carnitine by mitochondria, the oxygen uptake was consistent with the complete oxidation of the substrate. 2. The inhibition of β-oxidation by pent-4-enoate was partially reversed by very high concentrations of l-carnitine. Conditions were also defined for the sustained oxidation of pent-4-enoate by mitochondria. 3. Pent-4-enoate inhibited pyruvate oxidation, but only at concentrations much higher than those needed to inhibit β-oxidation. MCPA had no effect on either pyruvate or 2-oxoglutarate oxidation in mitochondria. 4. Soluble extracts of rat or ox liver mitochondria completely oxidized pent-4-enoyl-CoA and the oxidation of butyryl-CoA added subsequently was unaffected. Incubation of soluble extracts with pent-4-enoyl-CoA in the absence of cofactors caused inhibition of acetoacetyl-CoA thiolase activity. However, this enzyme was not inhibited in intact mitochondria by pent-4-enoate. 5. MCPA specifically inhibited butyryl-CoA dehydrogenase in both intact mitochondria and in soluble extracts supplemented with ATP and CoASH. 6. Both MCPP and MCPA (1 mM) caused a rapid decrease in CoASH concentrations in mitochondria; acetyl-CoA concentrations were unaffected. Concentrations of pent-4-enoate (20 μM) sufficient to inhibit β-oxidation caused only a slight decrease in CoASH whereas higher concentrations (0.1–1.0 mM) caused a more extensive depletion of CoASH. However, evidence is presented to suggest that CoASH depletion is not the mechanism by which these compounds inhibit β-oxidation. 7. Pent-4-enoate and MCPA were substrates for butyryl-CoA synthetase. K m and V max values for several unusual, straight and branched chain fatty acids were determined. 8. Some short-chain acyl-CoA esters were substrates for an acyl-CoA hydrolase located in the mitochondrial matrix. 9. Some short-chain acyl-CoA esters competitively inhibited the activation of pyruvate carboxylase by acetyl-CoA. 10. The possible mechanisms by which hypoglycin and pent-4-enoate cause inhibition of β-oxidation and hypoglycaemia in vivo are discussed.

The localization of hippurate synthesis in the matrix of rat liver mitochondria.

In many species benzoate is conjugated in the liver with glycine to form hippurate, after prior formation of benzoyl-CoA, which then reacts with glycine in a transacylation catalysed by glycine N-acylase (EC 2.3.1.13) (Bridges et al., 1970). This enzyme has been prepared from mitochondria (Schachter & Taggart, 1954; Bartlett & Gompertz, 1974), although there is no information about its submitochondrial localization. Conversion of benzoate into benzoyl-CoA is presumably catalysed by butyryl-CoA synthetase (EC 6.2.1.2) (Mahler et al., 1953), which is found in the mitochondrial matrix (Garland et al., 1970). If glycine N-acylase also occurs in the matrix both benzoate and glycine would have to enter this compartment, and the hippurate formed would have to leave. If it is outside the permeability barrier of the inner mitochondria1 membrane, activated benzoyl groups would have to be exported as benzoylcarnitine. We therefore investigated the submitochondrial localization of hippurate synthesis. The synthesis of hippurate by intact and by sonicated preparations of mitochondria was measured. Mitochondria were prepared as described by Osmundsen & Sherratt (1975) and incubated at 20°C in 120m~-KC1/2m~-MgSO~/5m~-Hepes[2-(N-2hydroxyethylpiperazine4'yl)sulphonic acid], adjusted to pH7.2 with solid KOH, with 50jm-[l-14C]benzoate and appropriately supplemented with lOmM-ATP, 0.5 ~M-COA and lorn-glycine. Antimycin (1 pg/ml) and oligomycin (I pg/ml) were also added to block ATP synthesis and hydrolysis respectively. Incubations were quenched with HClO, to give a final concentration of 3% (v/v). Samples were then chromatographed on Whatman SG 81 paper with diethyl ether/80% (v/v) formic acid (lO:l, v/v) (Osmundsen, 1973) and the separated benzoic acid, hippuric acid and benzoyl-CoA were detected with U.V. light (Rp values 0,0.80 and 0.95 respectively). These compounds were determined from measurements of the radioactivity of appropriate areas of the chromatograms. Benzoyl-CoA was synthesized by standard methods (Stadtman, 1957). More hippurate was synthesized by sonicated than by intact mitochondria (10 and 1.4nmol/mg of protein during a 1 h incubation, respectively). Added CoA was required for high rates of hippurate synthesis by sonicated mitochondria. Benzoyl-CoA synthetase activity was latent in intact mitochondria and was released by sonication, confirming that it is located in the matrix. Mitochondria treated with digitonin as described by Schnaitman & Greenwalt (1968) to remove the outer membrane and the contents of the intermembrane space, and suspended in lWmM-Tris/HCI, pH8.2 at 20°C, still synthesized hippurate (3.7nmol/mg of protein during a 1 h incubation). These observations indicate that glycine N-acylase occurs in the matrix, where it will have access to benzoyl-CoA generated in this compartment. Suspensions of sonicated mitochondria incubated with 5Ophi-benzoate attained a nearly constant concentration of benzoyl-CoA after lOmin (about 2 5 ~ ) . This fell when glycine was added, with the synthesis of more than an equivalent amount of hippurate. The concentration of benzoate also fell. The failure to convert allthe benzoateadded into benzoyl-CoA in the absence of glycine suggests that there is hydrolysis of benzoyl-CoA. Indeed, benzoyl-CoA hydrolase activity with an apparent K,,, of low was found in the supernatant fraction obtained after centrifugation of a suspension of sonicated mitochondria for 1 h at 150000g.,., although this activity was unstable. Further, short-chain acyl-CoA hydrolase activity occurs in the matrix of liver mitochondria (Osmundsen & Sherratt, 1975). When intact mitochondria were incubated with 5Op~-benzoate, the maximum extent of acylation of their CoA was only about 0.5nmol/mg of protein. Rat liver mitochondria contain about 2-3nmol of CoA/mg of protein (Holland & Sherratt, 1973), of which about 50% was acylated by deca-2,4,6,8-tetraenoate in the steady state

Radioassay of acetyl-CoA synthetase, propionyl-CoA synthetase and butyryl-CoA synthetase in brain

Simple radiochemical methods are described for the assay of acetyl-CoA synthetase, propionyl-CoA synthetase, and butyryl-CoA synthetase utilizing 14C-labeled acetate, propionate, and butyrate. Enzyme activity is followed by the measurement of incorporation of the 14C-labeled short-chain fatty acids into their CoA-derivatives. Separation of the 14C-labeled reactant from its reaction product is achieved by rapid chromatography of the reaction mixture on Dowex anion exchange columns. A method is described to correct for inactivation of the enzymes by sucrose.

The activation of short-chain fatty acids by the soluble fraction of guinea-pig heart and liver mitochondria the search for a distinct propionyl-CoA synthetase

1. 1. The ATP dependent acetyl-, propionyl- and butyryl-CoA synthetase activities were measured in the soluble fraction of both guinea-pig heart and liver mitochondria. 2. 2. When measured in 300 mM Tris-HCl, the V of propionate activation in heart ( = 892 munits/mg protein) is much higher than the V of acetate ( = 637 munits/mg protein) and butyrate activation ( = 143 munits/mg protein. Fatty acid competition experiments, however, clearly show that most of the propionate activation ( K m = 7.94 mM) is caused by the acetyl-CoA synthetase (EC 6.2.1.1) ( K m for acetate = 0.8 mM), while the remaining activity is probably caused by a butyryl-CoA synthetase ( k m for butyrate = 0.83 mM). This indicates that in guinea-pig heart the presence of a distinct propionyl-CoA synthetase is very unlikely. 3. 3. In liver a completely different pattern of short-chain fatty acid activation is found: low acetate activation and moderate propionate and butyrate activation. Substrate competition experiments and kinetics of fatty acid activation indicate that in this tissue a distinct propionyl-CoA synthetase is present with high affinity for propionate ( K m = 0.6 mM) and some affinity towards acetate and butyrate ( K m values respectively 11 mM and 5.4 mM).

Dietary changes of cytoplasmic acetyl-CoA synthetase in different rat tissues

1. 1. Cytoplasmic and mitochondrial acetyl-CoA synthetase (acetate: CoA ligase (AMP), EC 6.2.1.1) activities were determined in different organs of normal, fasted and carbohydrate-refed rats. In liver cytoplasm and epididymal fat tissue its activity decreased to about 50% of normal after fasting (impaired lipogenesis). Refeeding a 70% carbohydrate diet (induced lipogenesis) caused a rise to 170% of the activity in liver cytoplasm and a rise to 550% in epididymal fat tissue. Cytoplasmic acetyl-CoA synthetase activities of heart and kidney remained unaltered following fasting or carbohydrate refeeding. 2. 2. No change of mitochondrial acetyl-CoA synthetase activity could be observed in liver, kidney and heart. The different regulatory behaviour of cytoplasmic and mitochondrial acetyl-CoA synthetase activities in liver supports previous evidence of two different enzýme proteins. 3. 3. Propionate and butyrate activation were measured in cytoplasm and mitochondria of rat liver. From the response to different dietary regimens it is concluded that propionate is activated in cytoplasm mainly by acetyl-CoA synthetase and in mitochondria by butyryl-CoA synthetase (butyrate:CoA ligase (AMP), EC 6.2.1.a). 4. 4. The relevance of these findings to a possible function of cytoplasmic acetyl-CoA synthetase in the transfer of acetyl groups from mitochondria to cytoplasm is discussed.