Cloned (Comment) | Organism |
---|---|
stable expression of wild-type and mutant enzyme in Pichia pastoris, expression in Escherichia coli strain XL10 primarily in inclusion bodies, while the expression yields are lower in Pichia pastoris than in Escherichia coli, the purified enzyme is significantly more stable, the enzyme form has the same substrate specificity but is 150fold more active than the Escherichia coli-expressed enzyme | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
N117Q | site-directed mutagenesis, the mutant enzyme is less stable than the wild-type enzyme, while it shows similar catalytic properties and substrate specificity | Homo sapiens |
General Stability | Organism |
---|---|
the Escherichia coli-expressed enzyme form is quite unstable, losing a significant fraction of its catalytic activity within 24 h | Homo sapiens |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
N-acetylaspartic acid | substrate inhibition at concentration above 0.2 mM | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | the enzyme shows sigmoidal behaviour and cooperative substrate bindng at low substrate concentration of 0.05-0.2 mM | Homo sapiens | |
0.12 | - |
N-acetylaspartic acid | pH 7.2, recombinant Pichia pastoris-expressed enzyme | Homo sapiens | |
0.15 | - |
N-trifluoroacetylaspartic acid | pH 7.2, recombinant Pichia pastoris-expressed enzyme | Homo sapiens | |
0.21 | - |
N-trifluoroacetylaspartic acid | pH 7.2, recombinant Escherichia coli-expressed enzyme | Homo sapiens | |
0.36 | - |
N-acetylaspartic acid | pH 7.2, recombinant Escherichia coli-expressed enzyme | Homo sapiens |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | required for activity, spectroscopical determination of metal content, the enzyme contains about 1 Zn2+ per enzyme subunit | Homo sapiens |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
36000 | - |
2 * 36000, recombinant enzyme, SDS-PAGE | Homo sapiens |
73000 | - |
dynamic light scattering study | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-acetylaspartic acid + H2O | Homo sapiens | enzyme mutations cause the Canavan disease | L-asparatate + acetate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | - |
- |
- |
Oxidation Stability | Organism |
---|---|
the Pichia pastoris-expressed enzyme shows sensitivity to oxidation over time and will precipitate if not kept under the proper reducing conditions, addition of 1 mM DTT reverses the precipitated state of the enzyme with no significant loss of activity | Homo sapiens |
Posttranslational Modification | Comment | Organism |
---|---|---|
glycoprotein | carbohydrate determination by mass spectrometry, overview, the N-glycosylation site is N117 | Homo sapiens |
Purification (Comment) | Organism |
---|---|
recombinant enzyme by metal affinity chromatography, dialysis, and anion exchange chromatography | Homo sapiens |
Renatured (Comment) | Organism |
---|---|
the Pichia pastoris-expressed wild-type enzyme shows sensitivity to oxidation over time and will precipitate if not kept under the proper reducing conditions, addition of 1 mM DTT reverses the precipitated state of the enzyme with no significant loss of activity | Homo sapiens |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
brain | - |
Homo sapiens | - |
Storage Stability | Organism |
---|---|
4°C, purified wild-type enzyme, expressed | Homo sapiens |
the purified enzyme expressed in Pichia pastoris is significantly more stable than the Escherichia coli-expressed enzyme, losing less than 10% of its catalytic activity after 2 weeks when stored under conditions where the Escherichia coli-expressed enzyme becomes completely inactivated within 24 h, the Pichia pastoris-expressed enzyme shows sensitivity to oxidation over time and will precipitate if not kept under the proper reducing conditions | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-acetylaspartic acid + H2O | - |
Homo sapiens | L-asparatate + acetate | - |
? | |
N-acetylaspartic acid + H2O | enzyme mutations cause the Canavan disease | Homo sapiens | L-asparatate + acetate | - |
? | |
N-trifluoroacetyl-L-aspartate + H2O | - |
Homo sapiens | L-aspartate + trifluoroacetate | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 36000, recombinant enzyme, SDS-PAGE | Homo sapiens |
More | quarternary structure, dynamic light scattering | Homo sapiens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.083 | - |
N-acetylaspartic acid | pH 7.2, recombinant Escherichia coli-expressed enzyme | Homo sapiens | |
1.2 | - |
N-trifluoroacetylaspartic acid | pH 7.2, recombinant Escherichia coli-expressed enzyme | Homo sapiens | |
12.7 | - |
N-acetylaspartic acid | pH 7.2, recombinant Pichia pastoris-expressed enzyme | Homo sapiens | |
116 | - |
N-trifluoroacetylaspartic acid | pH 7.2, recombinant Pichia pastoris-expressed enzyme | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.2 | - |
assay at | Homo sapiens |