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Literature summary for 6.2.1.B11 extracted from

  • Jones, C.P.; Ingram-Smith, C.
    Biochemical and kinetic characterization of the recombinant ADP-forming acetyl coenzyme A synthetase from the amitochondriate protozoan Entamoeba histolytica (2014), Eukaryot. Cell, 13, 1530-1537 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain Rosetta2(DE3)pLysS Entamoeba histolytica

Inhibitors

Inhibitors Comment Organism Structure
acetyl phosphate
-
Entamoeba histolytica
AMP slight inhibition Entamoeba histolytica
ATP ATP inhibits the acetate-forming direction of the reaction, with 50% inhibitory concentration of 0.81 mM, which is in the range of its physiological concentration. Diphosphate displays mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. No inhibition by lactate and pyruvate Entamoeba histolytica
cAMP
-
Entamoeba histolytica
D-fructose 1,6-bisphosphate
-
Entamoeba histolytica
D-fructose 6-phosphate
-
Entamoeba histolytica
D-glucose 6-phosphate
-
Entamoeba histolytica
diphosphate diphosphate inhibits the acetate-forming direction of the reaction, with 50% inhibitory concentration of 0.75 mM, which is in the range of its physiological concentration. ATP displays mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate Entamoeba histolytica
glyoxylate inhibits the activity in the acetate-forming direction Entamoeba histolytica
NAD+
-
Entamoeba histolytica
NADH
-
Entamoeba histolytica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten steady-state kinetic analysis, pseudo-first-order reaction kinetic determinations are performed in both directions of the reaction Entamoeba histolytica
0.032
-
propionyl-CoA pH 7.3, 37°C, propionate-forming direction, with propionyl-CoA Entamoeba histolytica
0.04
-
acetyl-CoA pH 7.3, 37°C, acetate-forming direction Entamoeba histolytica
0.2
-
CoA pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
0.71
-
ADP pH 7.3, 37°C, propionate-forming direction, with propionyl-CoA Entamoeba histolytica
1.5
-
phosphate pH 7.3, 37°C, propionate-forming direction, with propionyl-CoA Entamoeba histolytica
1.6
-
ADP pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
1.6
-
CoA pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
1.8
-
phosphate pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
1.9
-
GDP pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
7.2
-
ATP pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
10
-
GTP pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
12
-
ATP pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
14
-
acetate pH 7.3, 37°C, acetyl-CoA-forming direction Entamoeba histolytica
29
-
propionate pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ can slightly substitute for Mg2+ Entamoeba histolytica
Co2+ can partially substitute for Mg2+ Entamoeba histolytica
Cu2+ can slightly substitute for Mg2+ Entamoeba histolytica
Mg2+ required for activity, best divalent cation Entamoeba histolytica
Mn2+ can substitute for Mg2+ Entamoeba histolytica
Ni2+ can slightly substitute for Mg2+ Entamoeba histolytica
Zn2+ can slightly substitute for Mg2+ Entamoeba histolytica

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
150000
-
recombinant His6-tagged enzyme, gel filtration Entamoeba histolytica

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + acetate + CoA Entamoeba histolytica
-
ADP + phosphate + acetyl-CoA
-
r
GTP + acetate + CoA Entamoeba histolytica
-
GDP + phosphate + acetyl-CoA
-
r

Organism

Organism UniProt Comment Textmining
Entamoeba histolytica Q9NAT4
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain Rosetta2(DE3)pLysS by nickel affinity chromatography Entamoeba histolytica

Source Tissue

Source Tissue Comment Organism Textmining
trophozoite
-
Entamoeba histolytica
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ADP + phosphate + acetyl-CoA
-
Entamoeba histolytica ATP + acetate + CoA
-
r
ADP + phosphate + propionyl-CoA
-
Entamoeba histolytica ATP + propionate + CoA
-
r
ATP + acetate + CoA
-
Entamoeba histolytica ADP + phosphate + acetyl-CoA
-
r
ATP + propionate + CoA
-
Entamoeba histolytica ADP + phosphate + propionyl-CoA
-
r
GDP + phosphate + acetyl-CoA
-
Entamoeba histolytica GTP + acetate + CoA
-
r
GTP + acetate + CoA
-
Entamoeba histolytica GDP + phosphate + acetyl-CoA
-
r
GTP + acetate + CoA GTP gives 57% of the activity with ATP Entamoeba histolytica GDP + phosphate + acetyl-CoA
-
r
ITP + acetate + CoA ITP gives 15% of the activity with ATP Entamoeba histolytica IDP + phosphate + acetyl-CoA
-
r
additional information the enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. Less than 4% of maximal activity is observed with isobutyrate, valerate, isovalerate, hexanoate, heptanoate, octanoate, succinate, or phenylacetate as the acyl substrate. Activity observed with ATP (representing 100% activity) is nearly double that with GTP (57%) and substantially higher than the activities observed with ITP (15%), CTP (4.1%), or UTP (1.6%), no activity is observed with TTP or diphosphate. Enzymatic activity in the acetate-forming direction is determined by measuring the release of CoA with a sulfhydryl group (CoASH) from acyl-CoA by using Ellman's thiol reagent (DTNB). Production of NTB2- by CoASH cleavage of DTNB is measured spectrophotometrically at 412 nm. Activity in the acetate-forming direction is confirmed using the hexokinase/glucose-6-phosphate dehydrogenase-coupled assay. This assay couples ATP formation to the reduction of NADP+ to NADPH, which is measured spectrophotometrically at 340 nm Entamoeba histolytica ?
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 78999, recombinant His6-tagged enzyme, sequence calculation and SDS-PAGE Entamoeba histolytica

Synonyms

Synonyms Comment Organism
ACD
-
Entamoeba histolytica
Acetyl-CoA synthetase (ADP-forming)
-
Entamoeba histolytica
ADP-forming acetyl coenzyme A synthetase
-
Entamoeba histolytica
EhACD
-
Entamoeba histolytica
More cf. EC 6.2.1.13 Entamoeba histolytica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
acetate-forming assay at Entamoeba histolytica
55
-
acetyl-CoA-forming direction Entamoeba histolytica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
18
-
phosphate pH 7.3, 37°C, acetate-forming direction, with propionyl-CoA Entamoeba histolytica
21
-
propionyl-CoA pH 7.3, 37°C, acetate-forming direction, with propionyl-CoA Entamoeba histolytica
24
-
ADP pH 7.3, 37°C, acetate-forming direction, with propionyl-CoA Entamoeba histolytica
110
-
acetyl-CoA pH 7.3, 37°C, acetate-forming direction Entamoeba histolytica
120
-
phosphate pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
140
-
ADP pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
170
-
GDP pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
180
-
GTP pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
190
-
propionate pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
220
-
CoA pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
240
-
acetate pH 7.3, 37°C, acetyl-CoA-forming direction Entamoeba histolytica
240
-
ATP pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
260
-
CoA pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
320
-
ATP pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.3
-
assay at Entamoeba histolytica

Cofactor

Cofactor Comment Organism Structure
ADP
-
Entamoeba histolytica
ATP
-
Entamoeba histolytica
GDP
-
Entamoeba histolytica
GTP
-
Entamoeba histolytica

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.81
-
pH 7.3, 37°C, acetate-forming direction Entamoeba histolytica ATP
0.83
-
pH 7.3, 37°C, acetate-forming direction Entamoeba histolytica diphosphate

General Information

General Information Comment Organism
physiological function enzyme ACD is regulated through inhibition by ATP and diphosphate in the acetate-forming direction Entamoeba histolytica

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.3
-
propionate pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
12
-
phosphate pH 7.3, 37°C, acetate-forming direction, with propionyl-CoA Entamoeba histolytica
16
-
acetate pH 7.3, 37°C, acetyl-CoA-forming direction Entamoeba histolytica
18
-
GTP pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
27
-
ATP pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
33
-
ATP pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
34
-
ADP pH 7.3, 37°C, acetate-forming direction, with propionyl-CoA Entamoeba histolytica
65
-
phosphate pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
89
-
ADP pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
90
-
GDP pH 7.3, 37°C, acetate-forming direction, with acetyl-CoA Entamoeba histolytica
160
-
CoA pH 7.3, 37°C, propionyl-CoA-forming direction, with propionate Entamoeba histolytica
650
-
propionyl-CoA pH 7.3, 37°C, acetate-forming direction, with propionyl-CoA Entamoeba histolytica
1100
-
CoA pH 7.3, 37°C, acetyl-CoA-forming direction, with acetate Entamoeba histolytica
2600
-
acetyl-CoA pH 7.3, 37°C, acetate-forming direction Entamoeba histolytica