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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)

The genes encoding the acetate-inducible enzyme acetyl-coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu-5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N-terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position. The facA transcribed sequence contains six introns: one in the long leader sequence, one in the 5' coding sequence not homologous with acu-5, and four within the sequence that is largely similar to that of acu-5. Only one intron, corresponding in size and position to the furthest downstream of the facA introns, is found in acu-5. The evolution of introns during the divergence of these two Ascomycete fungi is discussed. Each of the two genes has been transferred by transformation into the other species. Each species is evidently able to splice out the other's introns. Most transformants have normal acetate-induction of acetyl-CoA synthetase, implying that the two genes respond to transcriptional control signals common to both species, in spite of the striking divergence of their 5' ends.
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PMID:Comparison and cross-species expression of the acetyl-CoA synthetase genes of the Ascomycete fungi, Aspergillus nidulans and Neurospora crassa. 197 35

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80. The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM. At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed. The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S. cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition. The major difference between the two yeast strains was that S. cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid. The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase. The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed. This resulted in a reduction in yield and an increase in specific ethanol production. Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.
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PMID:Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. 197 65

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae. 256 99

In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.
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PMID:Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii. 257 8

1. Comparative data are presented of the activities of pyruvate dehydrogenase complex and acetyl-CoA synthetase and of the acetate content in homogenates from ventral grey matter in spinal cord from cows and two non-ruminant species, pigs and horses. The methods used in the study are evaluated and discussed. 2. The total pyruvate dehydrogenase complex activity was 24.9-29.9 mU/mg protein and did not differ between the species. The part of the complex that was in active form at the sampling occasion was 60, 85 and 95% in cows, pigs and horses, respectively. 3. Acetyl-CoA synthetase activity differed significantly between the species and was 0.93, 1.28 and 2.61 mU/mg protein in pigs, cows and horses, respectively. The highest cytosolic activity was found in the horses. Acetate concentration at half maximal reaction velocity (at saturating CoA and ATP levels) was found to be 0.15-0.70 mM and did not differ between the species. 4. Acetate content was 63, 83 and 96 micrograms/g wet wt in cows, horses and pigs, respectively. 5. It is concluded that there seems to be no striking difference in acetyl-CoA synthesis in peripheral nerves between ruminants and non-ruminant species.
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PMID:A comparative study on acetyl-CoA synthesising enzymes in spinal cord from cows, horses and pigs. 257 76

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.
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PMID:Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii. 285 85

Fast atom bombardment mass spectrometry (FAB-MS) has been used to measure positional isotope exchange rates in enzyme-catalyzed reactions. The technique has been applied to the reactions catalyzed by acetyl-CoA synthetase and argininosuccinate synthetase. The FAB technique is also able to quantitatively determine the oxygen-18 or oxygen-17 content of nucleotides on as little as 10 nmol of material with no prior derivatization. Acetyl-CoA synthetase has been shown by FAB-MS to catalyze the positional exchange of an oxygen-18 of ATP from the beta-nonbridge position to the alpha beta-bridge position in the presence of acetate. These results are consistent with acetyl adenylate as a reactive intermediate in this reaction. Argininosuccinate synthetase was shown not to catalyze a positional isotope exchange reaction designed to test for the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase was also found not to catalyze the transfer of oxygen-18 from [ureido-18O]citrulline to the alpha-phosphorus of ATP in the absence of added aspartate. This experiment was designed to test for the transient formation of carbodiimide as a reactive intermediate. These results suggest that either argininosuccinate synthetase does not catalyze the formation of citrulline adenylate or the enzyme is able to completely suppress the rotation of the phosphoryl groups of PPi.
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PMID:Measurement of positional isotope exchange rates in enzyme-catalyzed reactions by fast atom bombardment mass spectrometry: application to argininosuccinate synthetase. 286 75

The levels of various metabolites were measured in freeze-clamped samples of liver from triiodothyronine-treated and control rats to which either saline or ethanol (2.5 g/kg body weight) had been administered 2 hours earlier. It was found that ethanol led to a sharp increase in the hepatic acetate concentration in both hyperthyroid and euthyroid rats whereas lactate and pyruvate concentrations were lowered in both groups. The lactate/pyruvate ratio rose significantly in euthyroid animals that had received ethanol but the ratio remained relatively low in hyperthyroid rats. The adenine nucleotide phosphorylation potential, already low in hyperthyroid rats, was further lowered by ethanol. However, the most remarkable difference between the responses of euthyroid and hyperthyroid rats to ethanol was in the hepatic concentrations of ketone bodies, particularly 3-hydroxybutyrate. In control animals, administration of ethanol did not affect either the acetoacetate or 3-hydroxybutyrate concentration but, although the level of ketone bodies in the livers of hyperthyroid rats that had not received ethanol was the same as that of controls, there was a greater than fivefold increase in the 3-hydroxybutyrate level when ethanol was given. While this increase in ethanol-dependent ketogenesis is not explicable at this stage, hyperthyroidism did not increase the activity of cytoplasmic acetyl-CoA synthetase, an enzyme that is probably involved in the formation of ketone bodies from ethanol-derived acetate.
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PMID:Increased ketogenesis in hyperthyroid rats metabolizing ethanol. 286 28

The strain Aspergillus terreus IRRL 16043 can utilize glucose as well as acetate as a sole carbon source. Thirty-nine mutants were isolated from the wild-type by treatment with a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNTG) which could not utilize acetate as a sole carbon source, and were designated as acetate non-utilizing (acu). By complementation and biochemical analyses they were divided into three functional groups, acu A, acu B and acu C lacking isocitrate lyase, malate synthase and acetyl-CoA synthetase activity, respectively.
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PMID:Biochemical studies on acetate non-utilizing mutants of Aspergillus terreus IRRL 16043. 288 25

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.
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PMID:Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A. 289 70


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