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Literature summary extracted from

  • Braesen, C.; Schmidt, M.; Groetzinger, J.; Schoenheit, P.
    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 (2008), J. Biol. Chem., 283, 15409-15418.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.2.1.1 genes acdIa and acdIb encoding the alpha and beta subunits, expression in Escherichia coli strain BL21(DE3) Pyrococcus furiosus
6.2.1.13 acdIa and acdIb genes encoding alpha- and beta-subunit of ACD, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pyrococcus furiosus

Protein Variants

EC Number Protein Variants Comment Organism
6.2.1.1 D212Ebeta site-directed mutagenesis, the mutant shows 2-4% of wild-type activity, slightly impaired in arsenolysis Pyrococcus furiosus
6.2.1.1 D212Nbeta site-directed mutagenesis, the mutant shows highly reduced phosphorylation/phosphorolysis activity, but only slightly impaired in arsenolysis Pyrococcus furiosus
6.2.1.1 E218Qalpha site-directed mutagenesis, inactive mutant Pyrococcus furiosus
6.2.1.1 H257Dalpha site-directed mutagenesis, inactive mutant Pyrococcus furiosus
6.2.1.1 H71Abeta site-directed mutagenesis, inactive mutant concerning phosphorylation/phosphorolysis, slightly impaired in arsenolysis Pyrococcus furiosus
6.2.1.1 additional information all mutations in the alpha-subunit have dramatic effects on arsenolysis activity, while mutations in the beta-subunit cause only moderate loss of activity Pyrococcus furiosus
6.2.1.13 D212betaE site-directed mutagenesis, comparison of the wild-type CD spectrum with the mutant CD spectrum, structure and reaction kinetics, overview. The mutant shows 2-4% of the wild-type activity, phosphorylation of the mutant is reduced Pyrococcus furiosus
6.2.1.13 E218alphaD site-directed mutagenesis, comparison of the wild-type CD spectrum with the mutant CD spectrum, structure and reaction kinetics, overview. The mutant shows 1-10% of the wild-type activity, phopshorylation of the mutant is reduced Pyrococcus furiosus
6.2.1.13 E218alphaQ site-directed mutagenesis, comparison of the wild-type CD spectrum with the mutant CD spectrum, structure and reaction kinetics, overview. Inactive mutant, phopshorylation of the mutant is reduced Pyrococcus furiosus
6.2.1.13 E218Dalpha 1-10% of wild-type activity Pyrococcus furiosus
6.2.1.13 H257alphaD site-directed mutagenesis, comparison of the wild-type CD spectrum with the mutant CD spectrum, structure and reaction kinetics, overview. Inactive mutant, which is not phosphorylated at both the alpha- and beta-subunit Pyrococcus furiosus
6.2.1.13 H257Dalpha mutant shows no activity in either direction Pyrococcus furiosus
6.2.1.13 H71betaA site-directed mutagenesis, comparison of the wild-type CD spectrum with the mutant CD spectrum, structure and reaction kinetics, overview. Inactive mutant, which is impaired in phosphorylation of the beta subunit Pyrococcus furiosus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.1 additional information
-
 additional information kinetic parameters of wild type and mutant enzymes at 55°C, overview Pyrococcus furiosus
6.2.1.1 0.0037
-
 acetyl-CoA pH 7.5, 80°C, wild-type alpha-subunit Pyrococcus furiosus
6.2.1.1 0.0039
-
 acetyl-CoA pH 7.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.0042
-
 acetyl-CoA pH 7.5, 80°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.0139
-
 CoA pH 7.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.093
-
 ADP pH 7.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.221
-
 ATP pH 7.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.23
-
 phosphate pH 7.5, 80°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.272
-
 phosphate pH 7.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.1 0.625
-
 acetate pH 7.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 additional information
-
 additional information kinetics of wild-type and mutant enzymes Pyrococcus furiosus
6.2.1.13 0.0039
-
 acetyl-CoA pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.0062
-
 acetyl-CoA pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
6.2.1.13 0.0139
-
 CoA pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.014
-
 acetyl-CoA pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.041
-
 CoA pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
6.2.1.13 0.0741
-
 ADP pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
6.2.1.13 0.0771
-
 CoA pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
6.2.1.13 0.082
-
 acetyl-CoA pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
6.2.1.13 0.094
-
 ADP pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.221
-
 ATP pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.221
-
 ATP pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.272
-
 phosphate pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.272
-
 phosphate pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.283
-
 ADP pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
6.2.1.13 0.388
-
 ATP pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
6.2.1.13 0.473
-
 ATP pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
6.2.1.13 0.625
-
 acetate pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.625
-
 acetate pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
6.2.1.13 0.714
-
 phosphate pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
6.2.1.13 0.783
-
 acetate pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
6.2.1.13 0.794
-
 acetate pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
6.2.1.13 0.919
-
 phosphate pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.1 Mg2+
-
 Pyrococcus furiosus
6.2.1.13 Mg2+ required Pyrococcus furiosus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
6.2.1.13 25878
-
 2 * 49965 (alpha) + 2 * 25878 (beta), calculated from sequence Pyrococcus furiosus
6.2.1.13 27000
-
 2 * 47000, alpha-subunit, + 2 * 27000, beta-subunit, SDS-PAGE Pyrococcus furiosus
6.2.1.13 47000
-
 2 * 47000, alpha-subunit, + 2 * 27000, beta-subunit, SDS-PAGE Pyrococcus furiosus
6.2.1.13 49965
-
 2 * 49965 (alpha) + 2 * 25878 (beta), calculated from sequence Pyrococcus furiosus
6.2.1.13 150000
-
 gel filtration Pyrococcus furiosus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.2.1.1 ATP + acetate + CoA Pyrococcus furiosus
-
 AMP + diphosphate + acetyl-CoA
-
 ?
6.2.1.13 ATP + acetate + CoA Pyrococcus furiosus
-
 ADP + phosphate + acetyl-CoA
-
 ?
6.2.1.13 ATP + acetate + CoA Pyrococcus furiosus the enzyme catalyzes the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation ADP + phosphate + acetyl-CoA
-
 r

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.1 Pyrococcus furiosus
-
 genes acdIa and acdIb encoding the alpha and beta subunits
-
6.2.1.13 Pyrococcus furiosus
-
 ACD isoenzyme I, acdIa and acdIb genes encoding alpha- and beta-subunit of ACD
-
6.2.1.13 Pyrococcus furiosus E7FI45 and E7FHP1 E7FI45 (alpha-subunit), E7FHP1 (beta-subunit)
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
6.2.1.13 phosphoprotein His257alpha and His71beta are sites of transient phosphorylation Pyrococcus furiosus

Purification (Commentary)

EC Number Purification (Comment) Organism
6.2.1.1 recombinant alpha and beta subunits from Escherichia coli strain BL21(DE3) by heat tteatment at 90°C for 30 min, hydrophobic interaction chromatography, and gel filtration Pyrococcus furiosus
6.2.1.13 recombinant wild-type and mutant enzyme subunits from Escherichia coli strain BL21(DE3) by heat precipitation at 90°C for 30 min, followed by reconstitution of holoenzyme through hydrophobic interaction chromatography ultrafiltration, and gel filtration, the chromatographic steps are then repeated Pyrococcus furiosus
6.2.1.13 wild-type end mutant enzymes Pyrococcus furiosus

Reaction

EC Number Reaction Comment Organism Reaction ID
6.2.1.1 ATP + acetate + CoA = AMP + diphosphate + acetyl-CoA the ACD reaction follows a four-step mechanism including transient phosphorylation of two active site histidine residues, structure and reaction mechanism of ACD Pyrococcus furiosus
a
6.2.1.13 ATP + acetate + CoA = ADP + phosphate + acetyl-CoA reaction mechanism and structural modelling, formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His257alpha, overview Pyrococcus furiosus
a
6.2.1.13 ATP + acetate + CoA = ADP + phosphate + acetyl-CoA reaction mechanism: the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceeds in analogy to succinyl-CoA synthetases. In contrast to succinyl-CoA synthetases, in acetyl-CoA synthetase 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-71alpha. It is proposed that acetyl-CoA synthetase reaction follows a four-step mechanism including transient phosphorylation of two active site histidine residues Pyrococcus furiosus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.1 ADP + phosphate + acetyl-CoA
-
 Pyrococcus furiosus ATP + acetate + CoA
-
 ?
6.2.1.1 ATP + acetate + CoA
-
 Pyrococcus furiosus AMP + diphosphate + acetyl-CoA
-
 ?
6.2.1.1 ATP + acetate + CoA formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His257alpha, respectively. The phosphoryl group is transferred from the His257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His71beta Pyrococcus furiosus AMP + diphosphate + acetyl-CoA
-
 ir
6.2.1.1 additional information the enzyme performs arsenolysis, the alpha-subunit alone also catalyzes arsenolysis, overview Pyrococcus furiosus ?
-
 ?
6.2.1.13 ATP + acetate + CoA
-
 Pyrococcus furiosus ADP + phosphate + acetyl-CoA
-
 ?
6.2.1.13 ATP + acetate + CoA the enzyme catalyzes the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation Pyrococcus furiosus ADP + phosphate + acetyl-CoA
-
 r
6.2.1.13 ATP + acetate + CoA the phosphorolysis is catalyzed by the alpha-subunit alone, independent of the beta-subunit. Both the His257alpha and Glu218alpha are crucial for phosphorolysis. In case of Glu218alpha the charge is essential for activity and also the length of the side chain is important Pyrococcus furiosus ADP + phosphate + acetyl-CoA
-
 r
6.2.1.13 additional information the enzyme performs catalytic arsenolysis of acetyl-CoA Pyrococcus furiosus ?
-
 ?

Subunits

EC Number Subunits Comment Organism
6.2.1.1 More structural analysis by circular dichroism spectroscopy and structure modelling, comparison of domain organization of subunits to succinyl-CoA synthetases, EC 6.2.1.5, overview Pyrococcus furiosus
6.2.1.13 heterotetramer 2 * 49965 (alpha) + 2 * 25878 (beta), calculated from sequence Pyrococcus furiosus
6.2.1.13 More a heterotetrameric ACD with alpha2beta structure, structural model, ACD shows a spatial arrangement of the subunits different from Escherichia coli succinyl-CoA synthetase, SCS, but maintaining a similar catalytic site, structural consequences of the domain shuffling in ACD, overview Pyrococcus furiosus
6.2.1.13 tetramer 2 * 47000, alpha-subunit, + 2 * 27000, beta-subunit, SDS-PAGE Pyrococcus furiosus

Synonyms

EC Number Synonyms Comment Organism
6.2.1.1 ACD
-
 Pyrococcus furiosus
6.2.1.1 ADP-forming acetyl-CoA synthetase
-
 Pyrococcus furiosus
6.2.1.1 More ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases Pyrococcus furiosus
6.2.1.13 ACD
-
 Pyrococcus furiosus
6.2.1.13 ADP-forming acetyl-CoA synthetase
-
 Pyrococcus furiosus
6.2.1.13 PF1540 gene name alpha subunit Pyrococcus furiosus
6.2.1.13 PF1787 gene name, beta-subunit Pyrococcus furiosus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.2.1.1 55
-
 phosphorylation and phosphorolysis assays at Pyrococcus furiosus
6.2.1.1 80
-
 arsenolytic assay at and temperature optimum Pyrococcus furiosus
6.2.1.13 55
-
 assay at Pyrococcus furiosus
6.2.1.13 55
-
 assay at, both reaction directions Pyrococcus furiosus
6.2.1.13 80
-
 assay at, catalytic arsenolysis of acetyl-CoA Pyrococcus furiosus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.2.1.1 7.5
-
 assay at Pyrococcus furiosus
6.2.1.13 6.5
-
 assay at Pyrococcus furiosus
6.2.1.13 6.5
-
 assay at, both reaction directions Pyrococcus furiosus

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.1 ADP
-
 Pyrococcus furiosus
6.2.1.1 ATP
-
 Pyrococcus furiosus
6.2.1.13 ADP
-
 Pyrococcus furiosus
6.2.1.13 ATP
-
 Pyrococcus furiosus