Information on EC 3.1.1.56 - methylumbelliferyl-acetate deacetylase

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

EC NUMBER
COMMENTARY hide
3.1.1.56
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RECOMMENDED NAME
GeneOntology No.
methylumbelliferyl-acetate deacetylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-methylumbelliferyl acetate + H2O = 4-methylumbelliferone + acetate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of carboxylic ester
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SYSTEMATIC NAME
IUBMB Comments
4-methylumbelliferyl-acetate acylhydrolase
Acts on short-chain acyl esters of 4-methylumbelliferone, but not on naphthyl, indoxyl or thiocholine esters.
CAS REGISTRY NUMBER
COMMENTARY hide
83380-83-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
river water buffalo
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Manually annotated by BRENDA team
C57BL/6, female, healthy or with experimental autoimmune uveoretinitis (EAU)
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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an estD mutant is found to be susceptible to nitrite and to S-nitrosoglutathione. This mutant is also unable to infect and survive within human cervical epithelial cells, and it shows reduced ability to form a biofilm on these cells
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-naphthyl acetate + H2O
1-naphthol + acetate
show the reaction diagram
1-naphthyl butyrate + H2O
1-naphthol + butyrate
show the reaction diagram
2-naphthyl acetate + H2O
2-naphthol + acetate
show the reaction diagram
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?
4-methylumbelliferyl acetate + H2O
4-methylumbelliferone + acetate
show the reaction diagram
4-methylumbelliferyl butyrate + H2O
4-methylumbelliferone + butyrate
show the reaction diagram
4-methylumbelliferyl propionate + H2O
4-methylumbelliferone + propionate
show the reaction diagram
4-nitrophenyl acetate + H2O
4-nitrophenol + acetate
show the reaction diagram
4-nitrophenyl butyrate + H2O
4-nitrophenol + butyrate
show the reaction diagram
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-
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?
carboxyfluorescein diacetate + H2O
?
show the reaction diagram
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-
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?
naphthyl acetate + H2O
naphthol + acetate
show the reaction diagram
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?
p-nitrophenyl acetate + H2O
p-nitrophenol + acetate
show the reaction diagram
p-nitrophenyl butyrate + H2O
p-nitrophenol + butyrate
show the reaction diagram
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22°C, pH 7.4, no substrate inhibition below 1 mM p-nitrophenyl butyrate
quantification at 405 nm
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?
S-formylglutathione + H2O
formate + glutathione
show the reaction diagram
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-
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?
S-lactoylglutathione + H2O
lactone + glutathione
show the reaction diagram
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quantification at 240 nm
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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
4-methylumbelliferyl acetate + H2O
4-methylumbelliferone + acetate
show the reaction diagram
additional information
?
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the enzyme catalyses the hydrolysis of S-formylglutathione, a role in formaldehyde detoxification is possible
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CuSO4
cystamine
Dansylglutamylglycylarginyl chloromethyl ketone
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weak
Diethyl p-nitrophenyl phosphate
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at high concentration (22°C, pH 7.4), resistance to diethyl p-nitrophenyl phosphate (also paraoxon, an organophosphorus inhibitor) is mediated by W197
diisopropyl fluorophosphate
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dimethyl p-nitrophenyl phosphate
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also methyl paraoxon (an organophosphorus inhibitor), resistance not due to hydrolysis of inhibitor
HgCl2
iodoacetamide
oxidized glutathione
phenylmethylsulfonyl fluoride
thiol reactive compound
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additional information
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relatively insensitive to: 4-nitrophenyl phosphate and acetozolamide
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.86 - 3.1
1-naphthyl acetate
2.9 - 4
2-naphthyl acetate
0.067
4-Methylumbelliferyl butyrate
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pH 5.2, 37°C, isoenzymes Es D1 and Es D2
0.9
4-nitrophenyl acetate
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pH 5.2, 37°C, isoenzymes Es D1 and Es D2
0.55 - 0.83
4-nitrophenyl butyrate
0.01 - 0.076
4-yethylumbelliferyl acetate
0.056
carboxyfluorescin diacetate
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pH 7.0, 37°C
1.7
Naphthyl acetate
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pH 6.0, 23°C
0.013 - 2.6
p-nitrophenyl acetate
0.012 - 0.14
p-nitrophenyl butyrate
0.88
S-formylglutathione
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pH 7.0, 30°C
3
S-Lactoylglutathione
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mutant C60S/W197I; mutant W197I
additional information
p-nitrophenyl butyrate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0067 - 0.767
Diethyl p-nitrophenyl phosphate
0.00483 - 0.8
dimethyl p-nitrophenyl phosphate
0.0055 - 1.783
p-nitrophenyl acetate
0.0032 - 3.63
p-nitrophenyl butyrate
50 - 116.7
S-Lactoylglutathione
additional information
dimethyl p-nitrophenyl phosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
Diethyl p-nitrophenyl phosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 5.5
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methylumbelliferyl acetate, methylumbelliferyl butyrate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9.5
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pH 5: about 40% of activity maximum, pH 9.5: about 30% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Manually annotated by BRENDA team
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retinal autoantigen
Manually annotated by BRENDA team
additional information
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proteomic analysis of bone marrow and peripheral blood mononuclear cells in acute myeloid leukemia patients and healthy subjects
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
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isoenzyme Es D1 and Es D2, gel filtration
33930
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theoretical
34000
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1 * 34000, isoenzyme Es D1 and Es D2, SDS-PAGE
35000
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2 * 35000, isoenzyme Es D1 and Es D2, SDS-PAGE with or without 2-mercaptoethanol
40000
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gel filtration
56900
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to 62400 Da, dimer, size-exclusion chromatography
76000
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isoenzyme Es D1 and Es D2, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 34000, isoenzyme Es D1 and Es D2, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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isoenzymes contain very little if any carbohydrate
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop: 16°C, 3 days, reservoir solution: sodium citrate pH 5.6, 200 mM ammonium acetate, 30% (w/v) polyethylene glycol (PEG) 4000, crystal: space group P2(1), unit cell parameters: a: 51.54, b: 70.72, c: 65.01, alpha: 90°, beta: 108.84°, gamma: 90°, 2 molecules in the asymmetric unit, molecular replacement using PDB: 1PV1, co-crystallization and soaking with substrate failed, structure: several insertions in canonical alpha/beta-hydrolase fold, GxSxG motif, active site in positively charged cleft including residues S149, D226, and H260, and aromatic residues (H148, F172 W183, F228, H260, Y262) lined at the surface, catalysis mediated by residues S153, H264, and D230, oxyanion hole formed by e.g. L54 and M150
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apo-enzyme (PDB: 1PV1) and mutant W197I (PDB: 3C6B) following inhibition through phosphorylation with diethyl p-nitrophenyl phosphate, sitting drop: pH 4.6, 25.5% (w/v) polyethylene glycol (PEG) 4000, 15% glycerol, 17°C, crystals: space group: P2(1)2(1)2(1) for PDB: 1PV1 (four molecules in asymmetric unit, dimer of dimers), C2 for PDB: 3C6B, structure: PDB: 1PV1: three-layer sandwich without disulfide bonds, catalytic triad: S161, H276, D241 and adjacent C60, oxyanion hole: L58 and M162, PDB: 3C6B: tetrahedral shape, sulfenic acid at residue C60, structural superimposition of wild-type and mutant W197I: natural resistance to paraoxon (diethyl p-nitrophenyl phosphate, an organophosphorus inhibitor) due to steric hindrance by W197 within the enzyme’s acyl pocket (L58, W102, I189, F243, L248, W197)
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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15 min, about 25% loss of activity
45 - 80
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thermal denaturation, circular dichroism spectrophotometry, modification of mutant W197I with diethyl p-nitrophenyl phosphate increases melting temperature by 6°C
50
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15 min, about 40% loss of activity
70
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15 min, about 75% loss of activity
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
sensitive to oxidation, regulation by cysteine sulfenic acid (Cys60)
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691005
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by nickel-affinity chromatography (elution: 300 mM imidazole) followed by cleavage of His-tag by tobacco etch virus (TEV) protease, a second nickel-affinity chromatography, and size-exclusion chromatography (Superdex G75) in presence of 1 mM dithiothreitol
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isoenzyme Es D1 and Es D2
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partial, esterase D is identical to sialic acid-specific O-acetylesterase
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
from brain cDNA in pMCSG7 for inducible expression with N-terminal hexa-His-tag in Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D226A
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4.3% of wild-type activity
D226N
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9% of wild-type activity
H260A
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3% of wild-type activity
H260Q
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1.3% of wild-type activity
L54A
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83% of wild-type activity
M150
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162% of wild-type activity, possibly due to reduced steric hindrance
S149A
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catalytically inactive
S149T
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1.3% of wild-type activity
C60H/W197I
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increased kcat and similar KM for p-nitrophenyl substrates compared to mutant W197I, increased phoshorylation by diethyl p-nitrophenyl phosphate
C60K/W197I
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similar kcat and KM for p-nitrophenyl substrates compared to mutant W197I, increased phoshorylation by diethyl p-nitrophenyl phosphate, 11°C decreased melting temperature compared to mutant W197I
C60Q/W197I
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decreased catalytic activity, but no affect on KM for p-nitrophenyl acetate compared to mutant W197I
C60R/W197I
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similar kcat and KM for p-nitrophenyl substrates compared to mutant W197I, increased phoshorylation by diethyl p-nitrophenyl phosphate, 10°C decreased melting temperature compared to mutant W197I
C60S/W197I
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double mutant
G57H
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decreased catalytic activity, enhanced dephosphorylation rate, close to oxyanion hole
G57H/W197I
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catalytically inactive
L58H
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decreased catalytic activity, far from oxyanion hole
M162H
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similar catalytic activity like wild-type, far from oxyanion hole
M162H/C60S/W197I
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triple mutant
W197H
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comparable to mutant W197I
W197I
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p-nitrophenyl butyrate preferred over p-nitrophenyl acetate as substrate, increased specificity towards and turnover of p-nitrophenyl butyrate and S-lactoylglutathione, enhanced organophosphate inhibition under pseudo-first-order conditions, no enhanced organophosphate hydrolysis, similar spontaneous dephosphorylation rate like wild-type, deprotonated at residue C60 leads to formation of a sulfenic acid
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
medicine
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enzyme should serve as a genetic marker of retinoblstome
molecular biology