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D183N
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine. Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 13333fold lower than wild-type value. Biofilm-detachment activity is very low
E184Q
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine. Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 70.6fold lower than wild-type value. pH-optimum is shifted from pH 5.8 for wild-type enzyme to pH 6.8 for the mutant enzyme
E332Q
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine. Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 2000fold lower than wild-type value. Biofilm-detachment activity is 28fold less efficient than the wild-type
R27A
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine. Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 2400fold lower than wild-type value. Biofilm-detachment activity is 20fold less efficient than the wild-type
R27K
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine. Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 1714fold lower than wild-type value. Biofilm-detachment activity is 26fold less efficient than the wild-type
W237A
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine and 4-nitrophenyl N-acetyl-beta-D-glucosamine. Biofilm-detachment activity is 167fold less efficient than the wild-type
W330Y
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no activity with 4-methylumbelliferyl N-acetyl-beta-D-glucosamine and 4-nitrophenyl N-acetyl-beta-D-glucosamine. Biofilm-detachment activity is 19.2fold less efficient than the wild-type
Y187A
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Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 40fold lower than wild-type value. Kcat/KM for 4-methylumbelliferyl N-acetyl-beta-D-glucosamine is 4.9fold lower than wild-type value. Biofilm-detachment activity is 8.3fold less efficient than the wild-type
Y278A
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Kcat/KM for 4-nitrophenyl N-acetyl-beta-D-glucosamine is 923fold lower than wild-type value. Kcat/KM for 4-methylumbelliferyl N-acetyl-beta-D-glucosamine is 45fold lower than wild-type value. Biofilm-detachment activity is 11fold less efficient than the wild-type
betaAsp452Asn/betaLeu453Arg
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the mutant enzyme of HexB exhibits more than 30fold increase in its ability to hydrolyze a 6-sulfated substrate and is able to hydrolyze GM2 ganglioside when the GM2 activator protein is replaced by sodium taurocholate
D148A
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the mutant shows 868fold decreased catalytic efficiency with 3-fluoro-4-nitrophenyl N-acetyl-beta-D-galactosaminide compared to the wild type enzyme
D148N
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the mutant shows 4190fold decreased catalytic efficiency with 3-fluoro-4-nitrophenyl N-acetyl-beta-D-galactosaminide compared to the wild type enzyme
E149A
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the mutant shows 1.6fold decreased catalytic efficiency with 4-nitrophenyl N-acetyl-beta-D-galactosaminide and 0.85fold increased catalytic efficiency with 3-fluoro-4-nitrophenyl N-acetyl-beta-D-galactosaminide compared to the wild type enzyme
E149Q
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the mutant shows 2.2fold decreased catalytic efficiency with 4-nitrophenyl N-acetyl-beta-D-galactosaminide compared to the wild type enzyme
H92A
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the mutant shows 69fold decreased catalytic efficiency with 4-nitrophenyl N-acetyl-beta-D-galactosaminide compared to the wild type enzyme
R178H
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from patient with the genetic disease GM2-gangliosidosis B1 variant, mutated Hex A, altered active site of alpha subunit
R424Q
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the mutant form of HexA shows about 3fold increase in its Km-value for the complex of GM2 activator protein and GM2 ganglioside
E328A
activity similar to wild-type
E328Q
about 20% decrease in activity
H433A
about 20% decrease in activity
V327G
about 30% decrease in activity
W448A
mutation results in a more than 1000fold loss in enzyme activity, due mainly to the effect on kcat
W448F
about 50% decrease in activity
W490A
mutation leads to a 2277fold decrease in sensitivity toward TMG-chitotriomycin as well as an 18fold decrease in binding affinity for the substrate (GlcNAc)2
D321E
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site-directed mutagenesis, the Hex1 mutant shows reduced activity compared to the wild-type enzyme
D321N
-
site-directed mutagenesis, almost inactive Hex1 mutant
E322D
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site-directed mutagenesis, the Hex1 mutant shows highly reduced activity compared to the wild-type enzyme
E322Q
-
site-directed mutagenesis, the Hex1 mutant shows reduced activity compared to the wild-type enzyme
D306A
the mutant enzyme is inactive with 4-nitrophenyl N-acetyl-beta-D-glucosaminide and chitobiose
D306E
the mutant enzyme shows 4.2% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
D306E/E307D
the mutant enzyme shows 2.7% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
D306N
the mutant enzyme is inactive with 4-nitrophenyl N-acetyl-beta-D-glucosaminide and chitobiose
E307A
the mutant enzyme shows 2.0% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
E307D
the mutant enzyme shows 8.6% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
E307Q
the mutant enzyme shows 1.5% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
D306A
-
the mutant enzyme is inactive with 4-nitrophenyl N-acetyl-beta-D-glucosaminide and chitobiose
-
D306E
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the mutant enzyme shows 4.2% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
-
D306N
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the mutant enzyme is inactive with 4-nitrophenyl N-acetyl-beta-D-glucosaminide and chitobiose
-
E307A
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the mutant enzyme shows 2.0% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
-
E307D
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the mutant enzyme shows 8.6% activity with 4-nitrophenyl N-acetyl-beta-D-glucosaminide compared to the wild type and is inactive with chitobiose
-
W443A
mutant of catalytic domain GH20-1, comparable Km but much lower kcat values than wild-type, enzymatic activities kcat/Km of about 1% towards 4-nitrophenyl-N-acetylglucosamine and one-fourth towards N-acetylglucosaminyl-beta-1,2-Man
W443A/W876A
significant decrease in activity
W443A/W876A/Y482A/Y914A
no detectable activity
W443A/Y482A
mutant of catalytic domain GH20-1, no detectable activity
W443F
mutant of catalytic domain GH20-1, one-sixth of wild-type activity towards 4-nitrophenyl-N-acetylglucosamine
W876A
mutant of catalytic domain GH20-2, one-third of wild-type activity towards 4-nitrophenyl-N-acetylglucosamine
W876A/Y914A
mutant of catalytic domain GH20-2, no detectable activity
W876F
mutant of catalytic domain GH20-2, one-eighth of wild-type activity towards 4-nitrophenyl-N-acetylglucosamine
Y482A/Y914A
complete loss of activity
Y482F
mutant of catalytic domain GH20-1, one-third of wild-type activity towards 4-nitrophenyl-N-acetylglucosamine
Y914A
mutant of catalytic domain GH20-2, increase in kcat value
Y914F
mutant of catalytic domain GH20-2, activity comparable to wild-type
Y482A
mutant of catalytic domain GH20-1, 2fold increase in Km and one-third of kcat value, leading to a much lower activity towards 4-nitrophenyl-N-acetylglucosamine than wild-type. Mutation completely abolishes the enzymatic activity towards N-acetylglucosamine-beta-1,2-Man
Y482A
mutant of catalytic domain GH20-1, one-sixth of wild-type activity towards 4-nitrophenyl-N-acetylglucosamine, no activity with N-acetylglucosaminyl-beta-1,2-Man
additional information
construction of truncated enzyme mutants, GH84C fragments comprising the catalytic module only, or additionally the adjacent CBM32 or the FN3 modules
additional information
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construction of truncated enzyme mutants, GH84C fragments comprising the catalytic module only, or additionally the adjacent CBM32 or the FN3 modules
additional information
HEXB mutation, a 91 base pair deletion constituting the entirety of exon 12. 15 base pair deletion at the 3' end of intron 11
additional information
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HEXB mutation, a 91 base pair deletion constituting the entirety of exon 12. 15 base pair deletion at the 3' end of intron 11
additional information
-
construction of a C-terminally truncated TS12 beta-HEX1 mutant, Hex1-DELTAC, comprising amino acids 1-502. Hex1-DELTAC consists of two domains, an N-terminal domain, residues 14-157, that consists of two long alpha-helices and seven beta-sheets, and a central catalytic domain, the catalytic domain is a typical (beta/alpha)8-barrel, a cyclic 8fold repeat of beta-strand/loop/alpha-helix units in which the beta-strands form the central 8-stranded beta-barrel
additional information
enzyme consists of a tandem repeat of two 53% sequence-identical catalytic domains designated as GH20-1 and GH20-2, respectively. Separate expression of the two domains, GH20-1 has an enzymatic activity of one-fourth compared to GH20-2