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Literature summary for 6.2.1.13 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
Search for more literature for 6.2.1.13

Cloned(Commentary)

Cloned (Comment) Organism
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

Protein Variants Comment Organism
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
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
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
E218Dalpha 1-10% of wild-type activity Pyrococcus furiosus
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
H257Dalpha mutant shows no activity in either direction Pyrococcus furiosus
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]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of wild-type and mutant enzymes Pyrococcus furiosus
0.0039
-
 acetyl-CoA pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
0.0062
-
 acetyl-CoA pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
0.0139
-
 CoA pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
0.014
-
 acetyl-CoA pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
0.041
-
 CoA pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
0.0741
-
 ADP pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
0.0771
-
 CoA pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
0.082
-
 acetyl-CoA pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
0.094
-
 ADP pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
0.221
-
 ATP pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
0.221
-
 ATP pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
0.272
-
 phosphate pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
0.272
-
 phosphate pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
0.283
-
 ADP pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
0.388
-
 ATP pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
0.473
-
 ATP pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
0.625
-
 acetate pH 6.5, 55°C, recombinant wild-type enzyme Pyrococcus furiosus
0.625
-
 acetate pH 6.5, 55°C, wild-type enzyme Pyrococcus furiosus
0.714
-
 phosphate pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
0.783
-
 acetate pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus
0.794
-
 acetate pH 6.5, 55°C, mutant enzyme E217Dalpha Pyrococcus furiosus
0.919
-
 phosphate pH 6.5, 55°C, mutant enzyme H257Dalpha Pyrococcus furiosus

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Pyrococcus furiosus

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + acetate + CoA Pyrococcus furiosus
-
 ADP + phosphate + acetyl-CoA
-
 ?
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

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

Posttranslational Modification

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

Purification (Commentary)

Purification (Comment) Organism
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
wild-type end mutant enzymes Pyrococcus furiosus

Reaction

Reaction Comment Organism Reaction ID
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
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + acetate + CoA
-
 Pyrococcus furiosus ADP + phosphate + acetyl-CoA
-
 ?
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
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
additional information the enzyme performs catalytic arsenolysis of acetyl-CoA Pyrococcus furiosus ?
-
 ?

Subunits

Subunits Comment Organism
heterotetramer 2 * 49965 (alpha) + 2 * 25878 (beta), calculated from sequence Pyrococcus furiosus
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
tetramer 2 * 47000, alpha-subunit, + 2 * 27000, beta-subunit, SDS-PAGE Pyrococcus furiosus

Synonyms

Synonyms Comment Organism
ACD
-
 Pyrococcus furiosus
ADP-forming acetyl-CoA synthetase
-
 Pyrococcus furiosus
PF1540 gene name alpha subunit Pyrococcus furiosus
PF1787 gene name, beta-subunit Pyrococcus furiosus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
55
-
 assay at Pyrococcus furiosus
55
-
 assay at, both reaction directions Pyrococcus furiosus
80
-
 assay at, catalytic arsenolysis of acetyl-CoA Pyrococcus furiosus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
 assay at Pyrococcus furiosus
6.5
-
 assay at, both reaction directions Pyrococcus furiosus

Cofactor

Cofactor Comment Organism Structure
ADP
-
 Pyrococcus furiosus
ATP
-
 Pyrococcus furiosus