Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.2.1.13 (acetyl-CoA synthetase)
 451 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of a new coenzyme A analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-CoA, suitable for immobilisation through its terminal amino group to support matrices, is described. The synthetic route starts with bis(CoA) and involves the following steps: alkylation with iodoacetic acid and rearrangement yielding bis(N6-carboxymethyl-CoA), elongation of the carboxymethyl terminal with 1,6-diaminohexane using carbodiimide to yield bis(N6-[N-(6-aminohexyl)-carbamoylmethyl]-CoA) and finally the splitting of this bis[CoA analogue) through reduction with dithiothreitol to give the final product in approximately 10% overall yield. This CoA analogue showed 'coenzymic activity' with the enzymes acetyl-CoA synthetase, phosphotransacetylase and succinic thiokinase. Covalent binding of the CoA analogue to Sepharose 4B was normally carried out using its S-(5-thio-2-nitrobenzoic acid) derivative as this allows a convenient way for determining the amount of ligand coupled, based on the amount of 5-thio-2-nitrobenzoic acid liberated from the gel after reduction with dithiothreitol. After covalent binding of the CoA analogue to water-soluble activated dextran 70, the analogue was recycled while present in an ultrafiltration cell using the enzymes phosphotransacetylase and citrate synthase. The reaction was followed by measuring the citrate formed on addition of acetylphosphate and oxaloacetate. In affinity chromatographic studies it was shown that the CoA-Sepharose preparation could bind the CoA-dependent enzymes citrate synthase and succinic thiokinase and these could be biospecifically eluted using soluble CoA.
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PMID:N6-[N-(6-Aminohexyl)carbamoylmethyl]-coenzyme A. Synthesis and application in affinity chromatography and as an immobilized active coenzyme. 57 88

Methanobacterium thermoautotrophicum growing on H2 plus CO2 as sole carbon and energy source was found to contain acetate thiokinase (Acetyl CoA synthetase; EC 6.2.1.1); Acetate + ATP + CoA leads to Acetyl CoA + AMP + PPi. The apparent Km value for acetate was 40 microM. Acetate kinase (EC 2.7.2.1) and phosphotransacetylase (EC 2.3.1.8) could not be detected. The specific activity of acetate thiokinase was high in cells grown with limited H2 and CO2 supply (approximately 100 nmol/min . mg protein), it was low in exponentially grown cells (2 nmol/min . mg protein). This corresponded with the finding that cells growing linearly in the presence of acetate assimilated the monocarboxylic acid in high amounts (greater than 10% of the cell carbon was derived from acetate), whereas exponentially growing cells did not (less than 1% of cell carbon was derived from acetate). These latter observations indicated that acetate thiokinase and free acetate are not involved in autotrophic CO2 fixation in M. thermoautotrophicum. The presence and some kinetic properties of succinate thiokinase (EC 6.2.1.5), adenylate kinase (EC 2.7.4.3), and inorganic pyrophosphatase (EC 3.6.1.1) are also described.
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PMID:Acetate thiokinase and the assimilation of acetate in methanobacterium thermoautotrophicum. 611

The gene coding for the acetyl-CoA synthetase (ADP-forming) from the amitochondriate eukaryote Giardia lamblia has been expressed in Escherichia coli. The recombinant enzyme exhibited the same substrate specificity as the native enzyme, utilizing acetyl-CoA and adenine nucleotides as preferred substrates and less efficiently, propionyl- and succinyl-CoA. N- and C-terminal parts of the G. lamblia acetyl-CoA synthetase sequence were found to be homologous to the alpha- and beta-subunits, respectively, of succinyl-CoA synthetase. Sequence analysis of homologous enzymes from various bacteria, archaea, and the eukaryote, Plasmodium falciparum, identified conserved features in their organization, which allowed us to delineate a new superfamily of acyl-CoA synthetases (nucleoside diphosphate-forming) and its signature motifs. The representatives of this new superfamily of thiokinases vary in their domain arrangement, some consisting of separate alpha- and beta-subunits and others comprising fusion proteins in alpha-beta or beta-alpha orientation. The presence of homologs of acetyl-CoA synthetase (ADP-forming) in such human pathogens as G. lamblia, Yersinia pestis, Bordetella pertussis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhi, Porphyromonas gingivalis, and the malaria agent P. falciparum suggests that they might be used as potential drug targets.
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PMID:Acetyl-CoA synthetase from the amitochondriate eukaryote Giardia lamblia belongs to the newly recognized superfamily of acyl-CoA synthetases (Nucleoside diphosphate-forming). 1068 68

In this study, we used proteomics to better understand the growth on glucose of Escherichia coli in high cell density, fed-batch cultures and the response to overexpression of plasmid-encoded 6-phosphogluconolactonase (PGL). Using liquid chromatography coupled to electrospray mass spectrometry, at least 300 proteins were identified in the cytosolic fraction of the six time points used to monitor the fermentation. The relative abundance changes of selected proteins were obtained by comparing the peak area of the corresponding peptides at a particular m/z (mass over charge ratio) value. During the time course of samples collected during the rapid growth achieved under batch and fed-batch conditions, both the control and recombinant E. coli strains showed up-regulation of proteins participating in the tricarboxylic acid (TCA) cycle, particularly acetyl-CoA synthetase (AcCoAS), malate dehydrogenase (MDH), and succinyl-CoA synthetase (SuccCoAS). In the recombinant strain culture, fumarase was up-regulated until 35 h after inoculation but was not in the control strain culture. In addition, the proteomic measurement detected up-regulation of three well-characterized binding transport proteins in both control and recombinant strains. The up-regulation of TCA cycle enzymes is consistent with the increase in growth rate observed in the cell culture. In addition, up-regulation of these proteins demonstrated the importance of both the pentose-phosphate shunt and TCA cycle to the increased biosynthetic activity required by a high level protein synthesis. This study shows the potential of proteomics using shotgun sequencing (LC/MS of tryptic digests) to measure global changes in protein abundance during a fermentation process and will facilitate the development of robust manufacturing systems.
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PMID:Proteomic profiling of Escherichia coli proteins under high cell density fed-batch cultivation with overexpression of phosphogluconolactonase. 1620 43

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:
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PMID:Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue. 1837 46

Formation and excretion of acetate as a metabolic end product of energy metabolism occurs in many protist and helminth parasites, such as the parasitic helminths Fasciola hepatica, Haemonchus contortus and Ascaris suum, and the protist parasites, Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis as well as Trypanosoma and Leishmania spp. In all of these parasites acetate is a main end product of their energy metabolism, whereas acetate formation does not occur in their mammalian hosts. Acetate production might therefore harbour novel targets for the development of new anti-parasitic drugs. In parasites, acetate is produced from acetyl-CoA by two different reactions, both involving substrate level phosphorylation, that are catalysed by either a cytosolic acetyl-CoA synthetase (ACS) or an organellar acetate:succinate CoA-transferase (ASCT). The ACS reaction is directly coupled to ATP synthesis, whereas the ASCT reaction yields succinyl-CoA for ATP formation via succinyl-CoA synthetase (SCS). Based on recent work on the ASCTs of F. hepatica, T. vaginalis and Trypanosoma brucei we suggest the existence of three subfamilies of enzymes within the CoA-transferase family I. Enzymes of these three subfamilies catalyse the ASCT reaction in eukaryotes via the same mechanism, but the subfamilies share little sequence homology. The CoA-transferases of the three subfamilies are all present inside ATP-producing organelles of parasites, those of subfamily IA in the mitochondria of trypanosomatids, subfamily IB in the mitochondria of parasitic worms and subfamily IC in hydrogenosome-bearing parasites. Together with the recent characterisation among non-parasitic protists of yet a third route of acetate formation involving acetate kinase (ACK) and phosphotransacetylase (PTA) that was previously unknown among eukaryotes, these recent developments provide a good opportunity to have a closer look at eukaryotic acetate formation.
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PMID:Acetate formation in the energy metabolism of parasitic helminths and protists. 2008 67

ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. The related succinyl-CoA synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II.
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PMID:Investigating the mechanism of ADP-forming acetyl-CoA synthetase from the protozoan parasite Entamoeba histolytica. 2812 70