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Literature summary extracted from
- Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site (2015), eLife, 2015, e06181 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 3.1.3.1 | expressed in Escherichia coli SM547(DE3) cells | Escherichia coli |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 3.1.3.1 | mutant enzyme D101A/D153A, hanging drop vapor diffusion method, using 22% (w/v) PEG3350, 0.1 mM Bis-Tris, pH 5.0, 0.2 mM ammonium sulfate | Escherichia coli |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 3.1.3.1 | D101A | the mutant shows 64fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/D153A | the mutant shows 190fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/D153A/E322Y | the mutant shows 48000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/D153A/E322Y/K328A | the mutant shows 320000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/D153A/K328A | the mutant shows 5300fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/E322Y | the mutant shows 20000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/E322Y/K328A | the mutant shows 570000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/K328A | the mutant shows 23000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S | the mutant shows 11000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/D153A | the mutant shows 32000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/D153A/E322Y | the mutant shows 670000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/D153A/E322Y/K328A | the mutant shows 3700000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/D153A/K328A | the mutant shows 120000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/E322Y | the mutant shows 15000000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/E322Y/K328A | the mutant shows more than 30000000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D101A/R166S/K328A | the mutant shows 2300000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D153A | the mutant shows 230fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D153A/E322Y | the mutant shows 270000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D153A/E322Y/K328A | the mutant shows 2000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | D153A/K328A | the mutant shows 1400fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | E322Y/K328A | the mutant shows 420000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | E332Y | the mutant shows 88000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | K328A | the mutant shows 840fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S | the mutant shows 6300fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/D153A | the mutant shows 49000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/D153A/E322Y | the mutant shows 33000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/D153A/E322Y/K328A | the mutant shows 140000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/D153A/K328A | the mutant shows 180000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/E322Y | the mutant shows 39000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/E322Y/K328A | the mutant shows 1600000000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | R166S/K328A | the mutant shows 2600000fold decreased catalytic efficiency compared to the wild type enzyme | Escherichia coli |
| 3.1.3.1 | S102G/D101A/D153A/R166S/E322Y/K328A | inactive | Escherichia coli |
Inhibitors
| EC Number | Inhibitors | Comment | Organism | Structure |
|---|---|---|---|---|
| 3.1.3.1 | phosphate | - |
Escherichia coli |  |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.1.3.1 | 0.00036 | - |
4-nitrophenyl phosphate | wild type enzyme, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0005 | - |
4-nitrophenyl phosphate | mutant enzyme E332Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0021 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0022 | - |
4-nitrophenyl phosphate | mutant enzyme E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0024 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0026 | - |
4-nitrophenyl phosphate | mutant enzyme D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0036 | - |
4-nitrophenyl phosphate | mutant enzyme D101A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0048 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.005 | - |
4-nitrophenyl phosphate | mutant enzyme R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0053 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0054 | - |
4-nitrophenyl phosphate | mutant enzyme K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0062 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0084 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.011 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.012 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.027 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.052 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.063 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.078 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.14 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.14 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.15 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.16 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.22 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.35 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.44 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 3.1.3.1 | Mg2+ | required for activity | Escherichia coli | |
| 3.1.3.1 | Zn2+ | required for activity | Escherichia coli | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 3.1.3.1 | Escherichia coli | P00634 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 3.1.3.1 | amylose resin column chromatography | Escherichia coli |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.1.3.1 | 4-nitrophenyl phosphate + H2O | - |
Escherichia coli | 4-nitrophenol + phosphate | - |
? | |
| 3.1.3.1 | methyl phosphate + H2O | - |
Escherichia coli | methanol + phosphate | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 3.1.3.1 | homodimer | - |
Escherichia coli |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.1.3.1 | 0.000029 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.000057 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00024 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00032 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00073 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00089 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00096 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0014 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0026 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0034 | - |
4-nitrophenyl phosphate | mutant enzyme E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0083 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.016 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.061 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.07 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.083 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.5 | - |
4-nitrophenyl phosphate | mutant enzyme R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 1.1 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 1.6 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 2.2 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 3 | 6 | 4-nitrophenyl phosphate | mutant enzyme D101A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 3.4 | - |
4-nitrophenyl phosphate | mutant enzyme K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 4.2 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 4.6 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 7.6 | - |
4-nitrophenyl phosphate | mutant enzyme D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 12 | - |
4-nitrophenyl phosphate | wild type enzyme, at pH 8.0 and 25°C | Escherichia coli | |
kcat/KM [mM/s]
| EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 3.1.3.1 | 0.00002 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00017 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00039 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.00094 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0011 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0016 | - |
methyl phosphate | mutant enzyme E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0016 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.002 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0031 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.0044 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.011 | - |
methyl phosphate | mutant enzyme R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.013 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.019 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.061 | - |
methyl phosphate | mutant enzyme D101A/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.24 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.28 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.32 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 0.42 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 1.1 | - |
methyl phosphate | mutant enzyme D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 1.5 | - |
4-nitrophenyl phosphate | mutant enzyme E322Y/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 2.3 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/E322Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 2.7 | - |
methyl phosphate | mutant enzyme D101A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 3.6 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 5.5 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 7.2 | - |
4-nitrophenyl phosphate | mutant enzyme E332Y, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 13 | - |
4-nitrophenyl phosphate | mutant enzyme R166S/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 20 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 28 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 58 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 100 | - |
4-nitrophenyl phosphate | mutant enzyme R166S, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 120 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 330 | - |
4-nitrophenyl phosphate | mutant enzyme D101A/D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 440 | - |
4-nitrophenyl phosphate | mutant enzyme D153A/K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 750 | - |
4-nitrophenyl phosphate | mutant enzyme K328A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 1200 | - |
methyl phosphate | wild type enzyme, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 2800 | - |
4-nitrophenyl phosphate | mutant enzyme D153A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 9900 | - |
4-nitrophenyl phosphate | mutant enzyme D101A, at pH 8.0 and 25°C | Escherichia coli | |
| 3.1.3.1 | 33000 | - |
4-nitrophenyl phosphate | wild type enzyme, at pH 8.0 and 25°C | Escherichia coli | |
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