Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.2.1.1 (ACS)
78,556 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and Pi (acetyl-CoA + ADP + Pi --> acetate + ATP + CoA) [Schifer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an alpha2beta2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55 degrees C), the apparent Km values for acetyl-CoA, ADP and Pi were 17 microM, 60 microM and 200 microM, respectively. ADP and Pi could not be replaced by AMP and PPi, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent Vmax value in the direction of acetyl-CoA formation was about 40 U/mg (55 degrees C), and the apparent Km values for acetate, ATP and CoA were 660 microM, 80 microM and 30 microM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (100%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), butyrate (92%), isobutyrate (79%), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (<3%). In addition to ADP (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100 degrees C.
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PMID:Purification and properties of acetyl-CoA synthetase (ADP-forming), an archaeal enzyme of acetate formation and ATP synthesis, from the hyperthermophile Pyrococcus furiosus. 911 24

It is well established that extracellular choline is transported into central cholinergic nerve terminals by 'high' and 'low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-(14)C]acetate (0.1-100 microM) and the uptake of [1-(14)C]acetate and the amount of [14C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-(14)C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (Km) of 22 microM. The formation of [14C]ACh in the tissue as a function of extracellular [1-(14)C]acetate appeared to occur by both 'high' and 'low' affinity processes with apparent Km values of 0.5 and 19.6 microM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-(14)C]acetate uptake and the amount of [14C]ACh formed from that [1-(14)C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [14C]ACh formed from extracellular [1-(14)C]acetate. High K+ depolarization of hippocampal tissue loaded with extracellular [1-(14)C]acetate not only increased the synthesis but also the release of [14C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow.
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PMID:Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release. 912 30

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10-20 microM, assuming a stromal volume of 66 microl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 microM. Malonyl-CoA concentrations were also very low (<0.1-3.0 microM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 microM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA-acyl-carrier protein transacylase reactions at the concentrations of metabolites measured here are < 0.1-4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.
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PMID:Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase. 935 62

The superfamily of adenylate forming enzymes including peptide synthetases, acyl-CoA synthetases and insect luciferases is readily identified by the signature sequence SGTTGXPKG. This sequence including an invariant lysyl residue is located in a disordered loop region and was predicted to be of significant antigenicity. Antibodies were generated employing YTSGTTGRPKGC attached to bovine serum albumin and have been successfully used to identify respective enzymes and adenylate forming domains in multienzyme systems. These include the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetases of Aspergillus nidulans and Acremonium chrysogenum, gramicidin S synthetase 1 and tyrocidine synthetase 1 from Bacillus brevis, acetyl-CoA synthetase from Alcaligenes eutrophus and a putative peptide synthetase from Metarhizium anisopliae. Weaker or no reactions are observed when the amino acid in position X in the protein is non-basic or hydrophobic, which is respectively the case for gramicidin S synthetase 1 and luciferase.
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PMID:Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes. 953 May 7

The corrinoid iron-sulfur protein (CFeSP) from Clostridium thermoaceticum functions as a methyl carrier in the Wood-Ljungdahl pathway of acetyl-CoA synthesis. The small subunit (33 kDa) contains cobalt in a corrinoid cofactor, and the large subunit (55 kDa) contains a [4Fe-4S] cluster. The cobalt center is methylated by methyltetrahydrofolate (CH3-H4folate) to form a methylcobalt intermediate and, subsequently, is demethylated by carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). The work described here demonstrates that the [4Fe-4S] cluster is required to facilitate the reactivation of oxidatively inactivated Cob(II)amide to the active Co(I) state. Site-directed mutagenesis of the large subunit gene was used to change residue 20 from cysteine to alanine, which resulted in formation of a cluster with EPR and redox properties consistent with those of [3Fe-4S] clusters. The midpoint potential of the cluster in the C20A variant was approximately 500 mV more positive than that of the [4Fe-4S] cluster in the native enzyme. Accordingly, it was found that the Co center in the C20A mutant protein could be reduced artificially but was severely crippled in its ability to be reduced by physiological electron donors. This is probably because the reduced cluster of the C20A protein cannot provide the driving force needed to reduce Co(II) to Co(I), since the Co(II/I) midpoint potential is -504 mV. The C20A variant also was unable to catalyze the steady-state synthesis of acetyl-CoA when CH3-H4folate or methyl iodide were provided as methyl donors and CO and CODH/ACS as reductants. Addition of chemical reductants rescued the catalytically crippled variant form in both of these reactions. On the other hand, in single-turnover reactions, the methyl-Co state of the altered protein was fully active in methylating H4folate and in synthesizing acetyl-CoA in the presence of CO and CoA. The combined results strongly indicate that the FeS cluster of the CFeSP is necessary for reductive activation of Co(II) to Co(I) by physiological reductants but is not required for catalysis, e.g., demethylation of CH3-H4folate or methylation of CODH/ACS. We propose that, during reductive activation, electrons flow from the reduced electron-transfer protein (e.g., CODH/ACS or reduced ferredoxin (Fd)) to the FeS cluster which then directs electrons to the cobalt center for catalysis. These results also support earlier hypotheses that the methylation and demethylation reactions involving the CFeSP are SN2-type nucleophilic displacement reactions and do not involve radical chemistry.
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PMID:Role of the [4Fe-4S] cluster in reductive activation of the cobalt center of the corrinoid iron-sulfur protein from Clostridium thermoaceticum during acetate biosynthesis. 954 55

1. Formation of acyl-coenzymes (Co)A occurs as an obligatory step in the metabolism of a variety of endogenous substrates, including fatty acids. The reaction is catalysed by ATP-dependent acid:CoA ligases (EC 6.2.1.1-2.1.3; AMP forming), classified on the basis of their ability to conjugate saturated fatty acids of differing chain lengths, short (C2-C4), medium (C4-C12) and long (C10-C22). The enzymes are located in various cell compartments (cytosol, smooth endoplasmic reticulum, mitochondria and peroxisomes) and exhibit wide tissue distribution, with highest activity associated with liver and adipose tissue. 2. Formation of acyl-CoA is not unique to endogenous substrates, but also occurs as an obligatory step in the metabolism of some xenobiotic carboxylic acids. The mitochondrial medium-chain CoA ligase is principally associated with metabolism via amino acid conjugation and activates substrates such as benzoic and salicylic acids. Although amino acid conjugation was previously considered an a priori route of metabolism for xenobiotic-CoA, it is now recognized that these highly reactive and potentially toxic intermediates function as alternative substrates in pathways of intermediary metabolism, particularly those associated with lipid biosyntheses. 3. In addition to a role in fatty acid metabolism, the hepatic microsomal and peroxisomal long-chain-CoA-ligases have been implicated in the formation of the acyl-CoA thioesters of a variety of hypolipidaemic and peroxisome proliferating agents (e.g. clofibric acid) and of the R(-)-enantiomers of the commonly used 2-arylpropionic acid non-steroidal anti-inflammatory drugs (e.g. ibuprofen). In vitro kinetic studies using rat hepatic microsomes and peroxisomes have alluded to the possibility of xenobiotic-CoA ligase multiplicity. Although cDNA encoding a long-chain ligase have been isolated from rat and human liver, there is currently no molecular evidence of multiple isoforms. The gene has been localized to chromosome 4 and homology searches have revealed a significant similarity with enzymes of the luciferase family. 4. Increasing recognition that formation of a CoA conjugate increases chemical reactivity of xenobiotic carboxylic acids has led to an awareness that the relative activity, substrate specificity and intracellular location of the xenobiotic-CoA ligases may explain differences in toxicity. 5. Continued characterization of the human xenobiotic-CoA ligases in terms of substrate/inhibitor profiles and regulation, will allow a greater understanding of the role of these enzymes in the metabolism of carboxylic acids.
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PMID:Role of hepatic fatty acid:coenzyme A ligases in the metabolism of xenobiotic carboxylic acids. 978 15

Purified recombinant poly(hydroxyalkanoic acid), PHA, synthase from Chromatium vinosum was used to examine in vitro poly(3-hydroxybutyric acid) (P(3HB)) formation. In combination with purified propionyl-coenzyme A transferase of Clostridium propionicum a two-enzyme in vitro P(3HB) biosynthesis system was established which allowed the synthesis of P(3HB) from free D-(-)-3-hydroxybutyric acid as substrate. The coenzyme A residue for the activation of this hydroxyacid was provided by acetyl-coenzyme A. By adding acetyl-coenzyme A synthetase to this system, a three-enzyme in vitro P(3HB) biosynthesis system was established. Coenzyme A that was released during the polymerization reaction was coupled to acetate which again served as the coenzyme A donor for the activation of 3-hydroxybutyric acid. The energy for the in vitro P(3HB) synthesis was provided by ATP hydrolyses resulting in acetyl-coenzyme A synthesis catalyzed by the acetyl coenzyme A synthetase. In this way the in vitro synthesis of P(3HB) became independent of the consumption of the expensive coenzyme A. By this procedure a handy system is available to produce in vitro PHA on a semipreparative scale.
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PMID:In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system. 983 44

A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long acetyl-CoA synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.
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PMID:Cloning and sequencing of an acetyl-CoA synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia. 1037 39

Biochemical and genetic evidence is presented to demonstrate that the prpE gene of Salmonella typhimurium encodes propionyl-CoA synthetase, an enzyme required for the catabolism of propionate in this bacterium. While prpE mutants used propionate as carbon and energy source, prpE mutants that lacked acetyl-CoA synthetase (encoded by acs) did not, indicating that Acs can compensate for the lack of PrpE in prpE mutants. Cell-free extracts enriched for PrpE catalysed the formation of propionyl-CoA in a propionate-, ATP-, Mg2+- and HS-CoA dependent manner. Acetate substituted for propionate in the reaction at 48% the rate of propionate; butyrate was not a substrate for PrpE. The propionyl-CoA synthetase activity of PrpE was specific for ATP. GTP, ITP, CTP and TTP were not used as substrates by the enzyme. UV-visible spectrophotometry, HPLC and MS data demonstrated that propionyl-CoA was the product of the reaction catalysed by PrpE.
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PMID:The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. 1041 Dec 65

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [3H]glycerol, [3H]oleic acid (OA) or [3H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 microM), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [3H]OA into glycerolipids by 30-90%, and increased by about 60% the accumulation of free [3H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [3H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 microM, respectively; and in cytoplasm, 6 and 0.6 nmol/min/mg protein and 38 and 60 microM, respectively. Triacsin C (2-33 microM) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC50 and apparent Ki values were 13.5 microM and 14 microM in microsomes, and 3.8 microM and 4 microM in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.
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PMID:Heterogeneous long chain acyl-CoA synthetases control distribution of individual fatty acids in newly-formed glycerolipids of neuronal cells undergoing neurite outgrowth. 1044 57


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