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Studies on medium-chain fatty acyl-coenzyme a synthetase. Purification and properties.

ABSTRACT: 1. Medium-chain fatty acyl-CoA synthetase (EC 6.2.1.2) was isolated by the method of Mahler, Wakil & Bock (1953) and the enzyme activity determined by the disappearance of CoA in the presence either of butyrate and ATP or of butyryl-AMP, as well as by ATP formation from butyryl-AMP and PP(i). 2. Preincubation of the enzyme with CoA and ATP alone or together, followed by the removal of these substrates by gel filtration, caused a marked inhibition of ATP formation, contrary to results previously obtained with palmitoyl-CoA synthetase. 3. The effect of ATP on butyryl-AMP-dependent CoA disappearance was inconsistent. Low concentrations of ATP (0.1-0.5mm) always caused inhibition, whereas higher concentrations (5-10mm) activated in some enzyme preparations and inhibited in others. 4. This inconsistency was shown to be due to the presence of two enzyme fractions. Both fractions had similar activities when assayed by the butyryl-AMP- or butyrate-plus-ATP-dependent CoA disappearance. However, fraction I was activated by ATP as measured by butyryl-AMP-dependent CoA disappearance whereas fraction II was inhibited by it. Fraction I also catalysed ATP formation from butyryl-AMP and PP(i) whereas fraction II was lacking in such activity. 5. The relationship of these observations with respect to other known mechanisms of fatty acid-activating systems is discussed.

SUBMITTER: Bar-Tana J

PROVIDER: S-EPMC1186784 | biostudies-other | 1968 Sep

REPOSITORIES: biostudies-other

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Studies on medium-chain fatty acyl-coenzyme a synthetase. Enzyme fraction I: mechanism of reaction and allosteric properties.

Project description:1. The mechanism of butyrate activation catalysed by an enzyme fraction derived from ox liver particles (fraction I; Bar-Tana, Rose & Shapiro, 1968) was studied by an analysis of the initial-velocity pattern of the overall reaction and found to conform to the Bi Uni Uni Bi Ping Pong model (Cleland, 1963a,b,c) in agreement with the reaction scheme proposed by Berg (1956). 2. A homotropic co-operative effect was exerted by CoA on fraction I, whereas ATP and AMP functioned as heterotropic co-operative ligands with respect to butyryl-AMP-dependent CoA disappearance. On the other hand, PP(i) and butyryl-CoA showed antagonistic heterotropic effects when tested under similar conditions. With respect to the overall reaction CoA and ATP could be shown to function as co-operative homotropic modifiers. 3. Two interchangeable conformational states of the enzyme are therefore presumed to exist, state R, having a higher affinity for CoA and ATP and thus preferentially catalysing butyryl-AMP-dependent CoA disappearance (partial reaction b), and state T, favoured by the presence of PP(i), catalysing the formation of ATP from butyryl-AMP and PP(i) (partial reaction a) with greater efficiency. 4. These findings serve to explain the opposite effects of ATP on the partial reactions, as well as the inhibition by CoA and ATP of ATP formation (reaction a) and by PP(i) of the butyryl-AMP-dependent CoA disappearance (reaction b) (Bar-Tana et al. 1968). 5. The possible analogy of these observations to amino acid-activating and other similar systems is discussed.

| S-EPMC1186785 | biostudies-other

Studies on medium-chain fatty acyl-coenzyme a synthetase. Enzyme fraction II: mechanism of reaction and specific properties.

Project description:1. The mechanism of reaction of fatty acyl-CoA synthesis catalysed by fatty acyl-CoA synthetase from ox liver (fraction II; Bar-Tana, Rose & Shapiro, 1968) was investigated by a kinetic study of CoA disappearance dependent on butyrate plus ATP or butyryl-AMP (overall and partial reaction b respectively). 2. Contrary to findings with another enzyme (fraction I), a Bi Uni Uni Bi Ping Pong mechanism (Cleland, 1963a,b,c) corresponding to Berg's (1956) scheme of reaction was eliminated and an ordered Ter Ter mechanism with an A-C-B (standing for ATP, CoA and butyrate respectively) sequence of substrate entry for the overall reaction was established for fraction II. Partial reaction (b) was found to follow the ;Iso-Theorell-Chance' mechanism. 3. Also, in contrast with results obtained with fraction I, no allosteric properties could be demonstrated with fraction II.

| S-EPMC1186786 | biostudies-other

Characterization of an archaeal medium-chain acyl coenzyme A synthetase from Methanosarcina acetivorans.

Project description:Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated Macs(Ma), utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP(i) were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PP(i). These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The Macs(Ma) structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys(256), Cys(298), Gly(351), Trp(259), Trp(237), and Trp(254), were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.

| S-EPMC2976453 | biostudies-literature

Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.

Project description:FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6-12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

| S-EPMC4493641 | biostudies-literature

Long-chain acyl coenzyme A synthetase 1 overexpression in primary cultured Schwann cells prevents long chain fatty acid-induced oxidative stress and mitochondrial dysfunction.

Project description:AimsHigh circulating long chain fatty acids (LCFAs) are implicated in diabetic neuropathy (DN) development. Expression of the long-chain acyl-CoA synthetase 1 (Acsl1) gene, a gene required for LCFA metabolic activation, is altered in human and mouse diabetic peripheral nerve. We assessed the significance of Acsl1 upregulation in primary cultured Schwann cells.ResultsAcsl1 overexpression prevented oxidative stress (nitrotyrosine; hydroxyoctadecadienoic acids [HODEs]) and attenuated cellular injury (TUNEL) in Schwann cells following 12 h exposure to LCFAs (palmitate, linoleate, and oleate, 100 ?M). Acsl1 overexpression potentiated the observed increase in medium to long-chain acyl-carnitines following 12 h LCFA exposure. Data are consistent with increased mitochondrial LCFA uptake, largely directed to incomplete beta-oxidation. LCFAs uncoupled mitochondrial oxygen consumption from ATP production. Acsl1 overexpression corrected mitochondrial dysfunction, increasing coupling efficiency and decreasing proton leak.InnovationSchwann cell mitochondrial function is critical for peripheral nerve function, but research on Schwann cell mitochondrial dysfunction in response to hyperlipidemia is minimal. We demonstrate that high levels of a physiologically relevant mixture of LCFAs induce Schwann cell injury, but that improved mitochondrial uptake and metabolism attenuate this lipotoxicity.ConclusionAcsl1 overexpression improves Schwann cell function and survival following high LCFA exposure in vitro; however, the observed endogenous Acsl1 upregulation in peripheral nerve in response to diabetes is not sufficient to prevent the development of DN in murine models of DN. Therefore, targeted improvement in Schwann cell metabolic disposal of LCFAs may improve DN phenotypes.

| S-EPMC4086511 | biostudies-literature

The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism.

Project description:1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PP(i)-ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP(2)O(7) (2-) but not with the concentration of Mg(2+), as judged by PP(i)-ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PP(i). In PP(i)-ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PP(i)-ATP exchange. PP(i)-dATP exchange was less than PP(i)-ATP exchange at low concentrations of fatty acid, but at higher concentrations PP(i)-dATP exchange exceeded PP(i)-ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PP(i). The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PP(i). The inhibition by propionate was non-competitive with respect to propionyl-AMP and PP(i). 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PP(i)-ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PP(i) is probably the only product released.

| S-EPMC1166141 | biostudies-other

FadD from Pseudomonas putida CA-3 is a true long-chain fatty acyl coenzyme A synthetase that activates phenylalkanoic and alkanoic acids.

Project description:A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k(cat) and K(m) values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3DeltafadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3DeltafadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3DeltafadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longer-monomer units into the PHA polymer.

| S-EPMC2786608 | biostudies-literature

Loss of the Mitochondrial Fatty Acid ?-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function.

Project description:Medium-chain acyl-Coenzyme A dehydrogenase (MCAD) is involved in the initial step of mitochondrial fatty acid ?-oxidation (FAO). Loss of function results in MCAD deficiency, a disorder that usually presents in childhood with hypoketotic hypoglycemia, vomiting and lethargy. While the disruption of mitochondrial fatty acid metabolism is the primary metabolic defect, secondary defects in mitochondrial oxidative phosphorylation (OXPHOS) may also contribute to disease pathogenesis. Therefore, we examined OXPHOS activity and stability in MCAD-deficient patient fibroblasts that have no detectable MCAD protein. We found a deficit in mitochondrial oxygen consumption, with reduced steady-state levels of OXPHOS complexes I, III and IV, as well as the OXPHOS supercomplex. To examine the mechanisms involved, we generated an MCAD knockout (KO) using human 143B osteosarcoma cells. These cells also exhibited defects in OXPHOS complex function and steady-state levels, as well as disrupted biogenesis of newly-translated OXPHOS subunits. Overall, our findings suggest that the loss of MCAD is associated with a reduction in steady-state OXPHOS complex levels, resulting in secondary defects in OXPHOS function which may contribute to the pathology of MCAD deficiency.

| S-EPMC5760697 | biostudies-literature

Properties and substrate specificity of some reactions catalysed by a short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata.

Project description:1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.

| S-EPMC1166140 | biostudies-other

Plasmodium falciparum Sir2A preferentially hydrolyzes medium and long chain fatty acyl lysine.

Project description:Plasmodium falciparum Sir2A (PfSir2A), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, has been shown to regulate the expression of surface antigens to evade the detection by host immune surveillance. It is thought that PfSir2A achieves this by deacetylating histones. However, the deacetylase activity of PfSir2A is weak. Here we present enzymology and structural evidence supporting that PfSir2A catalyzes the hydrolysis of medium and long chain fatty acyl groups from lysine residues more efficiently. Furthermore, P. falciparum proteins are found to contain such fatty acyl lysine modifications that can be removed by purified PfSir2A in vitro. Together, the data suggest that the physiological function of PfSir2A in antigen variation may be achieved by removing medium and long chain fatty acyl groups from protein lysine residues. The robust activity of PfSir2A would also facilitate the development of PfSir2A inhibitors, which may have therapeutic value in malaria treatment.

| S-EPMC3262940 | biostudies-literature

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