Information on EC 3.2.1.22 - alpha-galactosidase and Organism(s) Homo sapiens

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Homo sapiens


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


The taxonomic range for the selected organisms is: Homo sapiens

EC NUMBER
COMMENTARY hide
3.2.1.22
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RECOMMENDED NAME
GeneOntology No.
alpha-galactosidase
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
melibiose degradation
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stachyose degradation
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metabolism of disaccharids
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Galactose metabolism
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Glycerolipid metabolism
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Sphingolipid metabolism
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Glycosphingolipid biosynthesis - globo and isoglobo series
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SYSTEMATIC NAME
IUBMB Comments
alpha-D-galactoside galactohydrolase
Also hydrolyses alpha-D-fucosides.
CAS REGISTRY NUMBER
COMMENTARY hide
9025-35-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-methylumbelliferyl alpha-D-galactoside + H2O
4-methylumbelliferone + alpha-D-galactose
show the reaction diagram
-
-
-
-
?
4-methylumbelliferyl alpha-D-galactoside + H2O
4-methylumbelliferone + D-galactose
show the reaction diagram
-
-
-
-
?
4-methylumbelliferyl alpha-D-galactoside + H2O
alpha-D-galactose + 4-methylumbelliferone
show the reaction diagram
-
-
-
-
?
4-methylumbelliferyl-alpha-D-galactoside + H2O
4-methylumbelliferol + alpha-D-galactose
show the reaction diagram
-
-
-
-
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4-nitrophenyl alpha-D-galactoside + H2O
4-nitrophenol + alpha-D-galactose
show the reaction diagram
-
-
-
-
?
galactobisylceramide + H2O
?
show the reaction diagram
-
-
-
-
?
globopentaose + H2O
?
show the reaction diagram
-
-
-
-
?
globotriaosylceramide + H2O
?
show the reaction diagram
-
-
-
-
?
globotriaosylceramide + H2O
D-Galalpha(1,4)D-Glu-ceramide + D-galactose
show the reaction diagram
-
i.e. D-Galalpha(1,4)D-Galalpha(1,4)D-Glu-ceramide
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-
?
globotriose + H2O
?
show the reaction diagram
-
-
-
-
?
globtriglycosylceramide + H2O
?
show the reaction diagram
-
-
-
-
?
melibiose + H2O
D-galactose + D-glucose
show the reaction diagram
-
-
-
-
?
o-nitrophenyl alpha-D-fucopyranoside + H2O
o-nitrophenol + D-fucose
show the reaction diagram
-
-
-
-
?
o-nitrophenyl-alpha-N-acetyl-galactosaminide + H2O
?
show the reaction diagram
-
-
-
-
?
trihexosylceramide + H2O
?
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
globotriaosylceramide + H2O
?
show the reaction diagram
-
-
-
-
?
globotriaosylceramide + H2O
D-Galalpha(1,4)D-Glu-ceramide + D-galactose
show the reaction diagram
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i.e. D-Galalpha(1,4)D-Galalpha(1,4)D-Glu-ceramide
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-
?
melibiose + H2O
D-galactose + D-glucose
show the reaction diagram
-
-
-
-
?
trihexosylceramide + H2O
?
show the reaction diagram
-
-
-
-
-
additional information
?
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-deoxy-L-altronojirimycin hydrochloride
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1000fold less effective than 1-deoxygalactonojirimycin
1-deoxygalactonojirimycin
galactostatin bisulfite
myo-inositol
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N-acetyl-D-galactosamine
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n-butyldeoxygalactonojirimycin
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2000fold less effective than 1-deoxygalactonojirimycin
sodium taurocholate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-deoxygalactonojirimycin
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.9 - 6.8
4-methylumbelliferyl alpha-D-galactopyranoside
2.2 - 4.5
4-methylumbelliferyl alpha-D-galactoside
1.8 - 16.3
4-methylumbelliferyl-alpha-D-galactoside
0.28
Galabiosylceramide
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3.7
globopentaose
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9.1
globotriose
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-
0.18
globtriglycosylceramide
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-
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8.8
o-nitrophenyl alpha-D-fucopyranoside
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1.3
o-nitrophenyl-alpha-N-acetyl-galactosaminide
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alpha-galactosidase B
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.038 - 0.047
1-deoxygalactonojirimycin
0.095 - 0.122
galactostatin bisulfite
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000048 - 0.000063
1-deoxygalactonojirimycin
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.052
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6.56
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17.6
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74.62
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purified recombinant enzyme
229000
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plasma alpha-galactosidase A
2060000
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placental alpha-galactosidase A
4070000
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spleen alpha-galactosidase A
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 4.5
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4.2
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wild-type and mutant enzymes S65T, S65A and S65D
4.3
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4-methylumbelliferyl-alpha-D-galactopyranoside as substrate
4.8
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o-nitrophenyl-alpha-N-acetyl-galactosaminide as substrate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 7.5
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wild type enzyme
3.5 - 6.5
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mutant enzymes
3.6 - 5.4
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pH 3.6: about 75% of maximal activity, pH 5.4: about 40% of maximal activity, wild-type enzyme and mutant enzymes S65T, S65A and E66D
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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enzyme activity is detected in unstimulated whole saliva and mainly due to isoform A activity. Activity is higher in unclarified samples than in clarified ones and shows wide daily variations. Activity in whole salivea is significantly higher than in glandular saliva
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
49800
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alpha-galactosidase A, 2 * 49800, SDS-PAGE
50000
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ER and Golgi enzyme forms, SDS-PAGE
96600
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gel filtration
101000
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gel filtration
103000
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gel filtration, alpha-galactosidase A
117000
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gel filtration, alpha-galactosidase B
150000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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x-ray crystallography
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of structural models of mutant enzyme responsible for Fabry disease and calculation of indexes. Structural changes in the classic Fabry disease group are generally large and tend to be in the core region of a protein or located in the functionally important region, including the active-site pocket. Structural changes in the variant Fabry disease group are small or localized on the surface of the molecule far away from the activte site. Structural changes due to amino acid substitutions for which substrate analogues are effective for improving the stability or transportation are small or localized on the molecular surface
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hanging drop vapor diffusion method, using 25% (w/v) PEG 4000, 200 mM (NH4)2SO4, and 100 mM NaCH3COO, pH 4.6, at 20C
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6
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37C, 20 min, mutant enzymes S65T, S65A and E66D are stable
654330
4 - 7
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37C, 20 min, wild-type enzyme is stable
654330
6.1
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mutant enzyme S65T, more than 50% loss of activity after 20 min above
654330
6.5
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mutant enzyme S65A, more than 50% loss of activity after 20 min above
654330
6.9
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mutant enzyme S66D, more than 50% loss of activity after 20 min above
654330
7
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20 min,mutant enzyme S65T shows 3% residual activity, mutant enzyme S65A shows 24% residual activity, mutant enzyme S66D shows 45% residual activity
654330
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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mutant enzymes Q279E and R301Q are almost completely inactivated after 30 min at 50C, while the wild-type enzyme retains 43% of its original activity even after 60 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, pH 6, 1 mg/ml human serum albumin or 5% polyvinylpyrolidone, less than 3% per month loss in activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
efficient and rapid purification procedure for recombinant alpha-galactosidase A by sequential Concanavalin A-Sepharose and immobilized thio-alpha-galactoside agarose column chromatography. This procedure is especially useful for the purification of mutant forms of alpha-galactosidase A, which are not stable under conventional purification techniques. Purification of the intracellular mutant enzyme M279I, expressed in COS-7 cells within 6 h at 62% overall yield
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Ni2+-Sepharose column chromatography
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recombinant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned to an improved baculovirus vector and expressed in insect cells at optimized growth conditions, the purified enzyme is taken up by Fabry fibroblasts in culture resulting in normal enzyme levels
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Cos-cells
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expressed in Escherichia coli and in Methylotrophic yeast Ogataea minuta TK5-3/Ura cells
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expressed in Trichoplusia ni insect cells
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expression in COS-7 cell
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expression in murine fibroblast
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expression in Pichia pastoris. Purified recombinant enzyme is taken up by fibroblasts dericed from fabry disease patients and normal enzyme levels can be restored under theses conditions
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expression of mutant enzyme S65T, S65A and E66D by using an expression system with baculovirus/insect cells, expression in COS cells
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expression with Autographa californica nuclear polyhedrosis virus
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highly expression in transgenic mice
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mutant enzymes Q279E and R301Q are expressed in Sf9 cells
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mutants expressed in COS-7 cells
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overexpression in CHO cells
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overexpression in Sf9 cells infected with recombinant baculovirus, expression in COS-7 cells
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transgenic mouse expressing human mutant alpha-galactosidase R301Q in an endogenous enzyme deficient background is a biochemical animal model for studying active site specific chaperone therapy for Fabry disease. Oral administration of 1-deoxygalactonojirimycin, a competitive inhibitor of alpha-galactosidase A and an effective active-site specific chaperone for Fabry disease, at 0.05 mM in the drinking water of the mice for 2 weeks results in 13.8-, 3.3-, 3.9-, and 2.6fold increase in enzyme activities in the heart, kidney, spleen and liver, respectively
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme activity in Fabry disease patients is significantly lower than values measured for healthy controls
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A156V
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reduced activity
A20P
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reduced activity
A97V
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reduced activity
C142W
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mutation isolated in patient with Fabry disease. 1-Deoxygalactonojirimycin has no significant effect on catalytic activity
D170A
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the mutant protein lacks the active site nucleophile
D231G
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mutation isolated in patient with Fabry disease. Protein expression is similar to wild-type, but mutant has no catalytic activity. 1-Deoxygalactonojirimycin has no significant effect on catalytic activity
D246N
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the mutant shows about 37% activity compared to the wild type enzyme
D266N
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mutation isolated in patient with Fabry disease. 1-Deoxygalactonojirimycin has no significant effect on catalytic activity
E59K
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reduced activity
E66D
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KM-value and maximal velocity with 4-methylumbelliferyl alpha-D-galactoside as substrate are similar to the wild-type values. The stability of the mutant enzyme at neutral pH is reduced to 54% of the wild-type enzyme. The intracellular activity in COS1 cells is 68% of the wild-type activity. Intracellular activity in COS1 cells is enhanced to 80% of wild-type activity by cultivation of cells with 1-deoxygalactonojirimycin
L166V
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reduced activity
M296I
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reduced activity
M296V
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reduced activity
M42V
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mutation isolated in patient with Fabry disease. 1-Deoxygalactonojirimycin enhances mutant catalytic activity
M51I
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enzymological parameter values are almost the same as those of the wild-type GLA. Km (4-methylumbelliferyl alpha-D-galactoside) similar to wild-type. Mutant enzyme is unstable under neutral pH conditions and acidic ones. The effect of imino sugars including 1-deoxygalactonojirimycin and galactostatin bisulfite on the purified mutant enzyme is examined. The imino sugars improve the stability of the mutant enzyme under both neutral and acidic pH conditions. The results of surface plasmon resonance biosensor assaying suggests that the imino sugars retain their binding activity as to the mutant enzyme under both neutral and acidic pH conditions
M72V
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reduced activity
N215S
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reduced activity
R112C
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mutation isolated in patient with Fabry disease. 1-Deoxygalactonojirimycin enhances mutant catalytic activity
R112H
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reduced activity
R118C
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the mutant shows about 20% activity compared to the wild type enzyme
R118G
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the mutant shows about 38% activity compared to the wild type enzyme
R118H
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the mutant shows about 68% activity compared to the wild type enzyme
R118L
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the mutant shows about 52% activity compared to the wild type enzyme
R118P
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the mutant shows about 48% activity compared to the wild type enzyme
R118S
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the mutant shows about 78% activity compared to the wild type enzyme
R356W
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reduced activity
S126C
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the mutant shows about 3% activity compared to the wild type enzyme
S126G
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the mutant shows about 52% activity compared to the wild type enzyme
S126I
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the mutant shows about 38% activity compared to the wild type enzyme
S126N
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the mutant shows about 17% activity compared to the wild type enzyme
S126R
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the mutant shows about 20% activity compared to the wild type enzyme
S126T
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the mutant shows about 48% activity compared to the wild type enzyme
S297F
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mutation isolated in patient with Fabry disease. 1-Deoxygalactonojirimycin has no significant effect on catalytic activity
S65A
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KM-value and maximal velocity with 4-methylumbelliferyl alpha-D-galactoside as substrate are similar to the wild-type values. The stability of the mutant enzyme at neutral pH is reduced to 29% of the wild-type enzyme. The intracellular activity in COS1 cells is 26% of the wild-type activity. Intracellular activity in COS1 cells is enhanced to 44% of wild-type activity by cultivation of cells with 1-deoxygalactonojirimycin
S65T
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KM-value and maximal velocity with 4-methylumbelliferyl alpha-D-galactoside as substrate are similar to the wild-type values. The stability of the mutant enzyme at neutral pH is reduced to 4% of the wild-type enzyme. The intracellular activity in COS1 cells is 9% of the wild-type activity. Intracellular activity in COS1 cells is enhanced to 34% of wild-type activity by cultivation of cells with 1-deoxygalactonojirimycin
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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determinantion of alpha-galactosidase A activity in samples from patients with Fabry disease and healthy controls. Average enzyme activity in dried blood spot samples prepared using EDTA tubes is higher compared to those spotted directly irrespective of disease status
medicine