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Literature summary for 2.7.2.12 extracted from

  • Fowler, M.L.; Ingram-Smith, C.; Smith, K.S.
    Novel pyrophosphate-forming acetate kinase from the protist Entamoeba histolytica (2012), Eukaryot. Cell, 11, 1249-1256.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.7.2.12

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli as a His-tagged fusion protein Entamoeba histolytica

Protein Variants

Protein Variants Comment Organism
H117A mutant is inactive in the direction of acetyl phosphate formation. In the direction of acetate formation, alteration of the His117 results in a 16fold increase in the Km for acetyl phosphate and a 95fold decreased kcat, and for a 1500fold reduction in catalytic efficiency Entamoeba histolytica
H172A mutant is inactive in the direction of acetyl phosphate formation. The His172Ala mutant has a similar Km for acetyl phosphate as wild-type, the kcat value is reduced 970fold Entamoeba histolytica
I116A mutant shows no substantial change in the Km for acetate, mutant has a 26fold decreased kcat. In the direction of acetate formation, the Ile116Ala and Ile116Leu variants both display a mild increase in the Km for acetyl phosphate and decreased kcat, resulting in 22- to 27fold-decreased catalytic efficiencies, respectively Entamoeba histolytica
I116L mutant shows no substantial change in the Km for acetate. In the direction of acetate formation, the Ile116Ala and Ile116Leu variants both display a mild increase in the Km for acetyl phosphate and decreased kcat, resulting in 22- to 27fold-decreased catalytic efficiencies, respectively Entamoeba histolytica
T223G mutant shows significant activity in the acetate-forming direction of the assay but does not display activity in the acyl phosphate-forming direction with any acyl substrate Entamoeba histolytica
T223P mutant shows little effect on enzyme activity in the direction of acyl phosphate formation. The enzyme remains capable of utilizing substrates as long as hexanoate, and the Km and kcat values for each substrate are comparable to wild-type. In the direction of acetate formation, the Km for acetyl phosphate increases 4fold, the kcat also shows a 4fold increase, and thus the catalytic efficiency, kcat/Km, is unchanged Entamoeba histolytica
V87A mutant displays 16- to 20fold decreased turnover rates in the direction of acetate synthesis but no change in the Km for acetyl phosphate, and they are inactive in the acetyl phosphate-forming direction Entamoeba histolytica
V87G mutant displays 16- to 20fold decreased turnover rates in the direction of acetate synthesis but no change in the Km for acetyl phosphate, and they are inactive in the acetyl phosphate-forming direction Entamoeba histolytica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.5
-
 acetyl phosphate 37°C, pH 7, wild-type Entamoeba histolytica
0.51
-
 acetyl phosphate 37°C, pH 7, mutant V87G Entamoeba histolytica
0.53
-
 acetyl phosphate 37°C, pH 7, mutant V87A Entamoeba histolytica
0.69
-
 acetyl phosphate 37°C, pH 7, mutant H172A Entamoeba histolytica
1.6
-
 acetyl phosphate 37°C, pH 7, mutant I116A Entamoeba histolytica
1.9
-
 diphosphate co-substrate: hexanoate, 45°C, pH 7.5, mutant T223P Entamoeba histolytica
2
-
 acetyl phosphate 37°C, pH 7, mutant T223P Entamoeba histolytica
2.4
-
 acetyl phosphate 37°C, pH 7, mutant I116L Entamoeba histolytica
3
-
 diphosphate co-substrate: valerate, 45°C, pH 7.5, mutant T223P Entamoeba histolytica
3.2
-
 diphosphate co-substrate: butyrate, 37°C, pH 7.5, wild-type Entamoeba histolytica
3.6
-
 diphosphate co-substrate: acetyl phosphate, 37°C, pH 7.5, wild-type Entamoeba histolytica
4
-
 diphosphate co-substrate: propionate, 45°C, pH 7.5, mutant T223P Entamoeba histolytica
4.8
-
 diphosphate co-substrate: propionate, 37°C, pH 7.5, wild-type Entamoeba histolytica
4.9
-
 diphosphate co-substrate: butyrate, 45°C, pH 7.5, mutant T223P Entamoeba histolytica
6.3
-
 diphosphate co-substrate: acetate, 45°C, pH 7.5, mutant T223P Entamoeba histolytica
6.3
-
 diphosphate co-substrate: valerate, 37°C, pH 7.5, wild-type Entamoeba histolytica
8
-
 acetyl phosphate 37°C, pH 7, mutant H117A Entamoeba histolytica
16.3
-
 diphosphate co-substrate: hexanoate, 37°C, pH 7.5, wild-type Entamoeba histolytica
20.4
-
 hexanoate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
20.5
-
 hexanoate 45°C, pH 7.5, wild-type Entamoeba histolytica
41.6
-
 valerate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
48.9
-
 phosphate co-substrate: acetyl phosphate, 37°C, pH 7, wild-type Entamoeba histolytica
56.5
-
 Butyrate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
56.7
-
 valerate 45°C, pH 7.5, wild-type Entamoeba histolytica
75.6
-
 Butyrate 45°C, pH 7.5, wild-type Entamoeba histolytica
79.8
-
 propionate 45°C, pH 7.5, wild-type Entamoeba histolytica
98.7
-
 propionate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
106
-
 acetate 45°C, pH 7.5, wild-type Entamoeba histolytica
169
-
 acetate 45°C, pH 7.5, mutant T223P Entamoeba histolytica

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+
-
 Entamoeba histolytica
Mg2+
-
 Entamoeba histolytica
Mn2+ only weak activity Entamoeba histolytica

Organism

Organism UniProt Comment Textmining
Entamoeba histolytica
-
-
-

Purification (Commentary)

Purification (Comment) Organism
using Ni-NTA chromatography Entamoeba histolytica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
diphosphate + acetate
-
 Entamoeba histolytica phosphate + acetyl phosphate
-
 r
diphosphate + butyrate
-
 Entamoeba histolytica phosphate + butyryl phosphate
-
 ?
diphosphate + hexanoate
-
 Entamoeba histolytica ?
-
 ?
diphosphate + valerate
-
 Entamoeba histolytica phosphate + valeryl phosphate
-
 ?
additional information unlike other characterized ACKs, EhACK shows only diphosphate-dependent activity in the direction of acetyl phosphate formation. ATP does not serve as a phosphoryl donor Entamoeba histolytica ?
-
 ?
phosphate + acetyl phosphate
-
 Entamoeba histolytica diphosphate + acetate
-
 r
propionate + acetate
-
 Entamoeba histolytica phosphate + propionyl phosphate
-
 ?

Synonyms

Synonyms Comment Organism
EhACK
-
 Entamoeba histolytica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 acetate-forming direction Entamoeba histolytica
45
-
 acetyl phosphate-forming direction Entamoeba histolytica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.051
-
 hexanoate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.065
-
 hexanoate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.18
-
 Butyrate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.19
-
 valerate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.21
-
 valerate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.33
-
 Butyrate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.61
-
 propionate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
1.16
-
 propionate 45°C, pH 7.5, wild-type Entamoeba histolytica
1.3
-
 acetate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
1.76
-
 acetate 37°C, pH 7, wild-type Entamoeba histolytica
2
-
 acetyl phosphate 37°C, pH 7, mutant H172A Entamoeba histolytica
21
-
 acetyl phosphate 37°C, pH 7, mutant H117A Entamoeba histolytica
97
-
 acetyl phosphate 37°C, pH 7, mutant V87G Entamoeba histolytica
280
-
 acetyl phosphate 37°C, pH 7, mutant I116A Entamoeba histolytica
344
-
 acetyl phosphate 37°C, pH 7, mutant I116L Entamoeba histolytica
1218
-
 acetyl phosphate 37°C, pH 7, mutant V87A Entamoeba histolytica
1939
-
 acetyl phosphate 37°C, pH 7, wild-type Entamoeba histolytica
8333
-
 acetyl phosphate 37°C, pH 7, mutant T223P Entamoeba histolytica

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Entamoeba histolytica

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0025
-
 hexanoate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.0032
-
 Butyrate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.0032
-
 hexanoate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.0034
-
 valerate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.0043
-
 Butyrate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.0049
-
 valerate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.0062
-
 propionate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.0077
-
 acetate 45°C, pH 7.5, mutant T223P Entamoeba histolytica
0.015
-
 propionate 45°C, pH 7.5, wild-type Entamoeba histolytica
0.016
-
 acetate 45°C, pH 7.5, wild-type Entamoeba histolytica
3846
-
 acetyl phosphate 37°C, pH 7, wild-type Entamoeba histolytica