Information on EC 3.5.1.26 - N4-(beta-N-acetylglucosaminyl)-L-asparaginase

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

EC NUMBER
COMMENTARY hide
3.5.1.26
-
RECOMMENDED NAME
GeneOntology No.
N4-(beta-N-acetylglucosaminyl)-L-asparaginase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O = N-acetyl-beta-D-glucosaminylamine + L-aspartate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylic acid amide hydrolysis
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
L-asparagine degradation III (mammalian)
-
-
Other glycan degradation
-
-
SYSTEMATIC NAME
IUBMB Comments
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine amidohydrolase
Acts only on asparagine-oligosaccharides containing one amino acid, i.e., the asparagine has free alpha-amino and alpha-carboxyl groups [cf. EC 3.5.1.52, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase]
CAS REGISTRY NUMBER
COMMENTARY hide
9075-24-5
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
additional information
a surface loop blocks the catalytic center of the mature hydrolase, autoproteolysis is therefore required to remove this P loop and open up the hydrolase center. Structures of the precursor and the mature form are found in a single crystal, structure comparisons, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-L-beta-aspartamido-(2-acetamido)-1,2-dideoxy-beta-D-galactose + H2O
L-aspartate + NH3 + 2-acetamido-2-deoxy-beta-D-galactose
show the reaction diagram
-
low activity
-
?
1-L-beta-aspartamido-beta-D-galactose + H2O
L-aspartate + beta-D-galactose + NH3
show the reaction diagram
-
-
-
?
1-L-beta-aspartamido-beta-D-glucose + H2O
L-aspartate + beta-D-glucose + NH3
show the reaction diagram
-
high activity
-
?
1-L-beta-aspartamido-beta-D-mannose + H2O
L-aspartate + beta-D-mannose + NH3
show the reaction diagram
-
-
-
?
2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-beta-D-glucose + H2O
2-acetamido-1-amino-1,2-dideoxy-beta-D-glucose + L-aspartate
show the reaction diagram
2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose + H2O
?
show the reaction diagram
-
-
-
?
L-asparagine + H2O
L-aspartate + NH3
show the reaction diagram
L-aspartic acid beta-(7-amido-4-methylcoumarin) + H2O
7-amino-4-methylcoumarin + L-aspartate
show the reaction diagram
L-aspartic acid-b-7-amido-4-methylcoumarin + H2O
7-amino-4-methylcoumarin + L-aspartate
show the reaction diagram
-
-
-
-
?
L-aspartic acid-p-nitrolanilide + H2O
p-nitroaniline + L-aspartate
show the reaction diagram
-
-
-
?
N-[alpha-D-Man-(1-3)-alpha-D-Man-(1-6)-[alpha-D-Man-(1-2)-alpha-D-Man-(1-2)-alpha-D-Man-(1-3)]-beta-D-Man-(1-4)-beta-D-GlcNAc-(1-4)-beta-D-GlcNAc]-L-aspartate + H2O
alpha-D-Man-(1-3)-alpha-D-Man-(1-6)-[alpha-D-Man-(1-2)-alpha-D-Man-(1-2)-alpha-D-Man-(1-3)]-beta-D-Man-(1-4)-beta-D-GlcNAc-(1-4)-beta-D-GlcNAc + L-aspartate
show the reaction diagram
-
fast hydrolysis rate
-
?
N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine + H2O
2-acetamido-2-deoxy-beta-D-glucopyranosylamine + L-asparagine
show the reaction diagram
-
catalyzes the hydrolysis of the N-glycosylic bond
-
-
?
N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine + H2O
?
show the reaction diagram
-
catabolism of N-linked oligosaccharides
-
-
?
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O
2-acetamido-1-amino-1,2-dideoxy-beta-D-glucose + L-aspartate
show the reaction diagram
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O
N-acetyl-beta-D-glucosamine + L-aspartate
show the reaction diagram
-
-
-
-
?
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O
N-acetyl-beta-D-glucosaminylamine + L-aspartate
show the reaction diagram
N4-(beta-N-acetylglucosaminyl)-L-asparagine + H2O
2-acetamido-1-amino-1,2-dideoxy-beta-D-glucose + L-aspartate
show the reaction diagram
-
deficiency in the activity of human GA leads to a lysosomal storage disease aspartylglycosaminuria
-
-
?
N4-(beta-N-acetylglucosaminyl)-L-asparagine + H2O
N-acetyl-D-glucosamine + L-asparagine
show the reaction diagram
-
-
-
?
ovalbumin + H2O
L-aspartate + ?
show the reaction diagram
-
-
-
?
ovalbumin glycopeptide + H2O
L-aspartate + ?
show the reaction diagram
-
intermediate activity
-
?
ribonuclease B glycopeptide + H2O
L-aspartate + ?
show the reaction diagram
-
poor substrate, more activity with the pure glycopeptide
-
?
sialo-transferrin + H2O
L-aspartate + ?
show the reaction diagram
-
intermediate activity
-
?
Taka-amylase + H2O
L-aspartate + ?
show the reaction diagram
-
-
-
?
additional information
?
-
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
L-asparagine + H2O
L-aspartate + NH3
show the reaction diagram
N-[alpha-D-Man-(1-3)-alpha-D-Man-(1-6)-[alpha-D-Man-(1-2)-alpha-D-Man-(1-2)-alpha-D-Man-(1-3)]-beta-D-Man-(1-4)-beta-D-GlcNAc-(1-4)-beta-D-GlcNAc]-L-aspartate + H2O
alpha-D-Man-(1-3)-alpha-D-Man-(1-6)-[alpha-D-Man-(1-2)-alpha-D-Man-(1-2)-alpha-D-Man-(1-3)]-beta-D-Man-(1-4)-beta-D-GlcNAc-(1-4)-beta-D-GlcNAc + L-aspartate
show the reaction diagram
-
fast hydrolysis rate
-
?
N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine + H2O
?
show the reaction diagram
-
catabolism of N-linked oligosaccharides
-
-
?
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O
2-acetamido-1-amino-1,2-dideoxy-beta-D-glucose + L-aspartate
show the reaction diagram
N4-(beta-N-acetylglucosaminyl)-L-asparagine + H2O
2-acetamido-1-amino-1,2-dideoxy-beta-D-glucose + L-aspartate
show the reaction diagram
-
deficiency in the activity of human GA leads to a lysosomal storage disease aspartylglycosaminuria
-
-
?
additional information
?
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
90% inhibition at 0.1%, v/v
3-Hydroxybutanone
-
competitive inhibition, Ki: 4.1 mM
3-phosphono-DL-2-aminopropionic acid
-
phosphono transition state mimic, competitive inhibitor
-
5-Diazo-4-oxo-L-norvaline
acetate
-
slight inhibition at 3.2 mM
aspartic acid
-
40% inhibition at 1 mM
Aspartylaniline
-
competitive inhibition
Aspartylcyclohexylamine
-
competitive inhibition
EDTA
-
50% inhibition at 1 mM
glycine
L-2-bromosuccinic acid
-
competitive inhibitor
-
L-2-chlorosuccinic acid
-
competitive inhibitor
-
L-2-methylsuccinic acid
-
competitive inhibitor
-
L-asparagine
-
competitive inhibition, Ki: 0.454 mM
L-aspartic acid
-
competitive inhibitor
L-cysteic acid
-
sulfo transition state mimic, competitive inhibitor
N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)bromoacetamide
-
substrate analogue, noncompetitive inhibitor
-
N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)chloroacetamide
-
substrate analogue, noncompetitive inhibitor
-
N-acetylcysteine
N1-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)glycinamide
-
substrate analogue, noncompetitive inhibitor
-
p-chloromercuribenzoate
-
50% inhibition at 1 mM
succinic acid
-
competitive inhibitor
additional information
-
not inhibited by L-cysteine
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cystathionine
-
20% activation at 5 mM
isoleucine
-
20% activation at 5 mM
methionine
-
26% activation at 5 mM
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1 - 14
1-L-beta-aspartamido-(2-acetamido)-1,2-dideoxy-beta-D-galactose
0.05 - 1
1-L-beta-aspartamido-(2-acetamido)-1,2-dideoxy-beta-D-glucose
10.8
1-L-beta-aspartamido-beta-D-galactose
-
-
5.7
1-L-beta-aspartamido-beta-D-mannose
-
-
0.143 - 0.444
2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose
0.4 - 0.64
2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta-D-glycopyranosylamine
0.66 - 12.1
L-asparagine
0.09 - 2.5
N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine
0.77
ovalbumin
-
-
-
additional information
additional information
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00015 - 16
additional information
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.9
3-phosphono-DL-2-aminopropionic acid
-
pH 5.8, 37C
-
1
Cu2+
-
-
1
EDTA
-
-
2.7
L-2-bromosuccinic acid
-
pH 5.8, 37C
-
7.7
L-2-chlorosuccinic acid
-
pH 5.8, 37C
-
0.7
L-2-methylsuccinic acid
-
pH 5.8, 37C
-
0.454
L-asparagine
-
competitive inhibition
0.6
L-aspartic acid
-
pH 5.8, 37C
1.4
L-cysteic acid
-
pH 5.8, 37C
0.56
N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)bromoacetamide
-
pH 5.8, 37C
-
0.64
N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)chloroacetamide
-
pH 5.8, 37C
-
0.75
N1-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)glycinamide
-
pH 5.8, 37C
-
1
p-chloromercuribenzoate
-
-
5
succinic acid
-
pH 5.8, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.21
-
before heat treatment
0.24
-
in lymphocytes
0.34
-
-
1.55
-
-
4.12
-
-
6.6 - 6.8
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 6
5.5
-
sharp activity peak
5.8
-
assay at
7 - 9
-
-
7.6
-
-
7.7 - 9
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
-
optimal activity within
6.5 - 8.5
-
only gradual decrease in activity within
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 56
-
enhanced activity at 56C
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
AGA maturation, activation cleavage of the dimerized AGA precursors into the N-terminal alpha- and the C-terminal beta-subunits takes place in the endoplasmic reticulum
Manually annotated by BRENDA team
additional information
L15R causes aspartylglucosaminuria and affects translocation of aspartylglucosaminidase
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16000
-
alpha1, 1 * 18000 + beta1, 1 * 16000, SDS-PAGE
16500
2 * 20700 + 2 * 16500, calculated from amino acid sequence
19000
-
heavy-chain2, 2 * 25000 + light-chain2, 2 * 19000
20000
-
alpha1, 1 * 24000 + beta1, 1 * 20000, SDS-PAGE
20700
2 * 20700 + 2 * 16500, calculated from amino acid sequence
24600
-
alpha1, 1 * 24600 + beta1, 1 * 18000, SDS-PAGE and heat-inactivation
28000
-
alpha 1, 1 * 28000 + beta 1, 1 * 17000, SDS-PAGE; alpha2,beta2, 2 * 28000 + 2 * 17000, SDS-PAGE
30000
2 * 30000 + 2 * 18000, active enzyme, SDS-PAGE
36740
deduced polypeptide with the signal peptide cleaved off, calculated from amino acid sequence
38000
-
HPLC/Blue chromatography
47000
-
gel filtration
56000
-
SDS-PAGE without heat-inactivation
63000
-
gel filtration
65000
-
gel filtration
70000
-
disc electrophoresis, ultracentrifugation
76000
-
1 * 76000, SDS-PAGE
80000
-
non-denaturing PAGE
84000
precursor, SDS-PAGE
88000
-
non-denaturing PAGE
101000
780000
-
gel filtration, analytical PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
2 * 20700 + 2 * 16500, calculated from amino acid sequence; 2 * 30000 + 2 * 18000, active enzyme, SDS-PAGE
homodimer
monomer
tetramer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
phosphoprotein
-
phosphate on high-mannose type glycosyl groups
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D151N mutant AGA precursor, complexed with glycine and without glycine
-
hanging drop vapor diffusion technique, 1.9 A resolution, precursor and autoprocessed enzymes
-
mutant with lower turnover number to be able to get a crystal with natural substrate in complex
-
purified recombinant mutant G172D enzyme precursor in presence of L-aspartic acid beta-hydroxamate, X-ray diffraction structure determination and analysis at 2.1 A resolution. Structures of the precursor and the mature form are found in a single crystal
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
45% activity
209066
6
-
stable above
209068
8.6
-
rapid fall in activity
209066
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
stable for 16 h at pH 7.6
65 - 80
65 - 70
-
denaturation
65 - 80
-
irreversible inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glycine inhibits autoproteolysis reaction
-
stabilized by its substrate Asn-GlcNAc
-
stable at higher pH values, stable in the presence of SDS
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 0.02 M phosphate buffer, pH 7.0, invariably stable
-
-20C, 1.5 years
-
-20C, 3 months
-
-20C, 30% glycerol, several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
near homogeneity
near homogeneity, 3step chromatography techniques
-
partial
recombinant wild-type and mutant enzymes, the latter as single-chain precursors
to homogeneity for liver enzyme
-
to homogeneity, chromatography techniques
to homogeneity, recombinant enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA sequence determination and analysis, overexpression of mutant L15R in BHK and COS-1 cells, low expression level in endoplasmic reticulum, the recombinant active enzyme does not reach the lysosomes
expression in COS cells
-
expression in COS-1 cells and in BHK-21 cells
expression in Escherichia coli
-
expression in NIH-3T3 cells
-
recombinant expression of wild-type and mutant enzymes
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D151N
-
mutation completely abolishes autoproteolysis, mutation eradicates the backbone distortion near the scissile peptide bond
T152A
-
autoproteolytically deficient, no hydrolase activity
T152C
-
mutant with lower turnover number to get a crystall in complex with natural substrate
G172D
the naturally occuring point mutation results in misprocessing of its precursor and is deficient in hydrolyzing glycoasparagines, the mutant can be stabilized by L-aspartic acid beta-hydroxamate for crystallization against proteolysis by other enzymes
T203I
the naturally occuring point mutation results in misprocessing of its precursor and is deficient in hydrolyzing glycoasparagines. The mutant shows increased thermostability but 93% reduced enzyme activity compared to the wild-type enzyme. The mutant is unstable to proteolysis by other enzymes
T152C
-
the autoproteolysis-active precursor is kinetically slower than the wild type enzyme
D200A
87% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
D201A
93% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
D205G
-
essential for activation by autocatalytic proteolytic processing
D70A
44% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
G226A
inactive mutant, study of folding, transport and catalytic kinetics of mutant AGA
G258A
inactive mutant, study of folding, transport and catalytic kinetics of mutant AGA
H124R
-
reduced dimerization of the precursor polypeptide, total secretion into the medium
H124W
-
reduced dimerization of the precursor polypeptide, total secretion into the medium
K230A
86% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
L15R
naturally occuring L15R, mutating the signal sequence, causes aspartylglucosaminuria and affects translocation of aspartylglucosaminidase
N225A
45% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
N308D
-
less than 10% of wild-type activity, misprocessing of precursor
N38D
-
30% of wild-type activity, proper processing to its mature lysosomal form
R234A
-
nearly complete decrease of activity, misprocessing of precursor in ER
R234L
-
nearly complete decrease of activity, misprocessing of precursor in ER
R234Q
-
nearly complete decrease of activity, misprocessing of precursor in ER
S238A
40% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
T224A
-
nearly complete decrease of activity
T224S
-
nearly complete decrease of activity
T257A
-
nearly complete decrease of activity; nearly complete decrease of activity, misprocessing of precursor in ER
T310A
-
less than 10% of wild-type activity, more properly cleaved form than N308D
T33A
48% of wild-type activity, study of folding, transport and catalytic kinetics of mutant AGA
T33S
same activity as wild-type AGA
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
medicine
Show AA Sequence (199 entries)
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