Information on EC 6.2.1.16 - acetoacetate-CoA ligase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
6.2.1.16
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RECOMMENDED NAME
GeneOntology No.
acetoacetate-CoA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + acetoacetate + CoA = AMP + diphosphate + acetoacetyl-CoA
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acid-thiol ligation
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Valine, leucine and isoleucine degradation
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Butanoate metabolism
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SYSTEMATIC NAME
IUBMB Comments
acetoacetate:CoA ligase (AMP-forming)
Also acts, more slowly, on L-3-hydroxybutanoate.
CAS REGISTRY NUMBER
COMMENTARY hide
39394-62-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
female, strain Sprague-Dawley
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
Zoogloea ramigera 1-16-M / ATCC 19623
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + acetate + CoA
AMP + diphosphate + acetyl-CoA
show the reaction diagram
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45% of the activity relative to acetoacetate
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-
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ATP + acetoacetate + CoA
AMP + diphosphate + acetoacetyl-CoA
show the reaction diagram
ATP + L-3-hydroxybutanoate + CoA
AMP + diphosphate + L-3-hydroxybutyryl-CoA
show the reaction diagram
ATP + L-3-hydroxybutyrate + CoA
AMP + diphosphate + L-3-hydroxybutyryl-CoA
show the reaction diagram
-
-
-
?
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
ATP + acetoacetate + CoA
AMP + diphosphate + acetoacetyl-CoA
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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can replace Mg2+ in activation, 26% of the activation relative to Mg2+
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AMP
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5 mM, 70% inhibition
diphosphate
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10 mM, 70% inhibition
phosphate
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physiological function
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hydrodynamics-based gene transduction shows that overexpression of AACS (1-547) increases the protein expression of caveolin-1, the principal component of the caveolae. Cleavage of AACS by legumain is critical for the regulation of enzymatic activity and results in gain-of-function changes
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additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Tris
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stimulates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.008 - 0.3
acetoacetate
0.033 - 0.06
ATP
0.01 - 0.091
CoA
1.4
L(+)-3-hydroxybutyrate
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-
0.075
L-(+)-3-hydroxybutyrate
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20
L-3-hydroxybutyrate
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pH 8.4
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.89
acetoacetate
pH 7.5, 30°C, recombinant enzyme
6.43
ATP
pH 7.5, 30°C, recombinant enzyme
8.16
CoA
pH 7.5, 30°C, recombinant enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
26.6
acetoacetate
pH 7.5, 30°C, recombinant enzyme
32.4
ATP
pH 7.5, 30°C, recombinant enzyme
23.6
CoA
pH 7.5, 30°C, recombinant enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00424
22°C, pH 8.0
4.91 - 6.8
purified recombinant enzyme, pH 7.5, 30°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7.4
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8.4
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activation of acetoacetate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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pH 6.0: about 30% of maximal activity, pH 9.0: about 65% of maximal activity
6.2 - 8.6
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about 65% of maximal activity at pH 6.2 and 8.6
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
in cerebral cortex expression of AACS mRNA is restricetd to neuronal cells
Manually annotated by BRENDA team
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low expression level
Manually annotated by BRENDA team
expression of AACS mRNA in the cerebellum is restricted primarily to glial cells
Manually annotated by BRENDA team
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very low expression level
Manually annotated by BRENDA team
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lactating
Manually annotated by BRENDA team
in cerebral cortex expression of AACS mRNA is restricetd to neuronal cells
Manually annotated by BRENDA team
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low expression level
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75143
x * 75143, deduced from DNA sequence
80000
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gel filtration
100000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 75143, deduced from DNA sequence
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetylation
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Streptomyces lividans GCN5-typeN-acetyltransferase SlPatA acetylates the enzyme at the active-site residue Lys617, the acetylation inactivates the enzyme, overview. Acetylated SlAacS is deacetylated by a sirtuin-type protein deacetylase. SlAacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl-CoA synthetase activity in all domains of life
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme SlAacS complexed with AMP and acetoacetate, hanging drop vapor diffusion method, mixing of 11 mg/ml protein and 1.5fold molar excess of buffered AMP and acetoacetate with a reservoir solution containing 0.7-1.2M K3citrate and 50 mM BTP-HCl, pH 7.0, 14°C, 1 week, X-ray diffraction structure determination and analysis, molecular replacement using full-length Salmonella enterica acetyl-CoA synthetase, PDB ID1PG4, stripped of waters and cofactors/substrates as search model
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
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1 d, 30% loss of activity, in absence of glycerol
30
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1 d, 70% loss of activity, in absence of glycerol
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
40% loss of activity on freezing and thawing without glycerol
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in presence of 10% glycerol the enzyme can be frozen and thawed ten times without loss of activity
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in presence of 5% glycerol the enzyme can be frozen and thawed five times without loss of activity
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 0.07 mg/ml enzyme, 10 mM Tris/HCl buffer, pH 7.5, 50% glycerol, 10 mM 2-mercaptoethanol, stable for at least 1 month
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-20°C, 0.1 mg/ml enzyme, 10 mM Tris-HCl, pH 7.5, 50% glycerol, 10 mM 2-mercaptoethanol, stable for at least 3 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by a method that includes His affinity resin chromatography
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recombinant enzyme
recombinant FLAG-tagged enzyme from Lenti-X-293T cells by affinity chromatography and ultrafiltration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
AACS DNa and amino acid sequence determination and analysis, and promoter identification and cloning, the human AACS promoter is a peroxisome-proliferator-activated receptor gamm, PPARgamma, target gene, the nuclear receptor is recruited to the AACS promoter by direct interaction with stimulating protein-1, Sp-1
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DNA and amino acid sequence determination, expression in Escherichia coli JM109, the engineered strain utilizes acetoacetate and can synthesize carotenoids, e.g. alpha-humulene, effectively, overview
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expressed in Escherichia coli
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expression of a cDNA isolated from human carcinoma HepG2 in Escherichia coli
gene AACS, analysis of the transcriptional mechanism of AACS in Neuro-2a neuroblastoma cells. The minimal core promoter of the mouse AACS gene is located in a region with 110 bps upstream from the transcription start site. Mutagenesis studies show that the Sp1 binding site is crucial for AACS promoter activity. Sp1 binds to the Sp1 binding site on the promoter region of AACS. Overexpression of Sp1 increases AACS mRNA levels. Real-time PCR enzyme expression analysis. Functional analysis of the 5'-flanking region of AACS in Neuro-2a cells
gene Aacs, cloning and recombinant expression of FLAG-tagged enzyme in Lenti-X-293T cells. AACS and legumain are transiently expressed in HEK-293 cells
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gene aacS, expression in Escherichia coli and complementation of a deficiency in atoDA, encoding acetyl-CoA:acetotacetate CoA transferase, EC 2.8.3.8, in Escherichia coli strain DELtaatoDA DELTAcobB DELTApka
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gene Aacs, recombinant expression of FLAG-tagged enzyme in Lenti-X-293T cells, overexpression of wild-type and mutant enzymes in HEK-293 cells
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recombinant enzyme expression
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
peroxisome-proliferator-activated receptor gamm, PPARgamma, induces AACS and adipogenesis. A PPAR-responsive element, PPRE-independent mechanism might be involved in PPARgamma-mediated AACS gene expression. PPARgamma-dependent activation of the human AACS gene is mediated by the GC boxes
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specificity protein 1 (Sp1) binds to the Sp1 binding site on the promoter region of AACS. Overexpression of Sp1 increases AACS mRNA levels
specificity protein 1 (Sp1) regulates gene expression of AACS in Neuro-2a cells, investigation of promoter activity of AACS, overview
the human AACS promoter is a PPARgamma target gene. Nuclear receptor PPARgamma is recruited to the AACS promoter by direct interaction with Sp1
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treatment of hepatocytes with U18666A results in the upregulation of AACS gene expression
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N320Q
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site-directed mutagenesis, the mutant is not cleaved by legumain
N449Q
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site-directed mutagenesis, the mutant is not cleaved by legumain
N500Q
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site-directed mutagenesis, the mutant is cleaved by legumain
N503Q
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site-directed mutagenesis, the mutant iscleaved by legumain, but the mutation abolishes the formation of the 56-kDa band through cleavage by legumain
N545Q
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site-directed mutagenesis, catalytically inactive mutant, that is cleaved by legumain
N547Q
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site-directed mutagenesis, the mutant iscleaved by legumain, but the mutation abolishes the formation of the 56-kDa band through cleavage by legumain
additional information
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