Information on EC 2.7.2.12 - acetate kinase (diphosphate)

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The expected taxonomic range for this enzyme is: Entamoeba histolytica

EC NUMBER
COMMENTARY hide
2.7.2.12
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RECOMMENDED NAME
GeneOntology No.
acetate kinase (diphosphate)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
diphosphate + acetate = phosphate + acetyl phosphate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
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Pyruvate metabolism
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SYSTEMATIC NAME
IUBMB Comments
diphosphate:acetate phosphotransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
57657-58-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain H200
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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residue Asn337 of ATP-ecoAK is particularly significant for the specificity to ATP. The five residues are highly conserved in 2625 PPi-ehiAK homologue implying that almost all organisms have ATP-dependent acetate kinase, EC 2.7.2.1, rather than diphosphate-dependent acetate kinase, EC 2.7.2.12
malfunction
AcK gene silencing does not affect acetate production in the parasites but promotes a slight decrease (10-20%) in the hexose phosphates and phosphate levels. Amoebae lacking ADP-forming acetyl-CoA synthetase activity are unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge
physiological function
The enzyme does not importantly contribute to ATP and phospate supply in the amoebae. Recombinant enzyme AcK shows activity only in the acetate formation direction. Enzyme AcK does not contribute to acetate production but might be marginally involved in phosphate and hexosephosphate homeostasis
additional information
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substrate-binding site structure and comparison with ATP-dependent acetate kinase, EC 2.7.2.1, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
diphosphate + acetate
phosphate + acetyl phosphate
show the reaction diagram
diphosphate + butyrate
phosphate + butyryl phosphate
show the reaction diagram
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-
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?
diphosphate + hexanoate
?
show the reaction diagram
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-
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?
diphosphate + valerate
phosphate + valeryl phosphate
show the reaction diagram
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-
-
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?
phosphate + acetyl phosphate
diphosphate + acetate
show the reaction diagram
propionate + acetate
phosphate + propionyl phosphate
show the reaction diagram
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-
-
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
diphosphate + acetate
phosphate + acetyl phosphate
show the reaction diagram
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-
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?
phosphate + acetyl phosphate
diphosphate + acetate
show the reaction diagram
C4M1C3
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r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
diphosphate
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purified enzyme PPi-ehiAK utilizes diphosphate, but not ATP, as a phosphoryl donor
additional information
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no activity with ATP
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MgCl2
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employed in assay mixture
Mn2+
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only weak activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
106 - 169
acetate
0.02 - 8
acetyl phosphate
56.5 - 75.6
Butyrate
1.9 - 16.3
diphosphate
20.4 - 20.5
hexanoate
2.2 - 48.9
phosphate
79.8 - 98.7
propionate
41.6 - 56.7
valerate
additional information
additional information
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Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 1.76
acetate
2 - 8333
acetyl phosphate
0.18 - 0.33
Butyrate
2.6
diphosphate
Entamoeba histolytica
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pH 6.5-7.5, 30C, recombinant enzyme
0.051 - 0.065
hexanoate
0.61 - 1.16
propionate
0.19 - 0.21
valerate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0077 - 0.016
acetate
47
300 - 3846
acetyl phosphate
358
0.0032 - 0.0043
Butyrate
462
0.0025 - 0.0032
hexanoate
1061
9 - 12
phosphate
16
0.0062 - 0.015
propionate
312
0.0034 - 0.0049
valerate
2040
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
diphosphate and acetate formation reaction, assay at
6.5 - 7.5
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acetyl phosphate-forming reaction direction, assay at
6.5
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assay at, diphosphate forming reaction direction
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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acetyl phosphate-forming direction
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 44000, recombinant His-tagged enzyme, SDS-PAGE
monomer
1 * 45000, about, sequence calculation, x * 77000, about, recombinant His6-tagged enzyme, SDS-PAGE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, under N2, stable for a week
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by cobalt affinity chromatography
using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli as a His-tagged fusion protein
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gene CL6EHI_170010, DNA and amino acid sequence determination and analysis, quantitative expression analysis by qRT-PCR, recombinant overexpression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
sequence comparisons, recombinant overexpression of His-tagged enzyme in Escherichia coli strain MK3648
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H117A
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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
H172A
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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
I116A
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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
I116L
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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
T223G
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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
T223P
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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
V87A
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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
V87G
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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
additional information