Cloned (Comment) | Organism |
---|---|
overexpression in Escherichia coli from pET28a plasmids | Pyrococcus furiosus |
Protein Variants | Comment | Organism |
---|---|---|
A408S | A408S mutation results in a significant increase in both dNTP binding affinity and fidelity, kcat/Km for dATP is about 45% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity | Pyrococcus furiosus |
D215A | 3'-5'-exonuclease inactive mutant enzyme | Pyrococcus furiosus |
L409I | kcat/Km for dATP is about 20% compared to the wild-type enzyme | Pyrococcus furiosus |
L409M | L409 mutation results in drastically reduced affinity for the correct dNTP, a much higher efficiency of both misincorporation and mismatch extension, and substantially lower fidelity as demonstrated by a PCR-based forward mutation assay, kcat/Km for dATP is about 155% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity | Pyrococcus furiosus |
L409V | kcat/Km for dATP is about 35% compared to the wild-type enzyme | Pyrococcus furiosus |
Y410I | kcat/Km for dATP is about 20% compared to the wild-type enzyme | Pyrococcus furiosus |
Y410L | kcat/Km for dATP is about 45% compared to the wild-type enzyme | Pyrococcus furiosus |
Y410V | Y410V mutation results in high fidelity in both misincorporation assays and forward mutation assays, but displays a substantially higher Km than wild-type enzyme, kcat/Km for dATP is about 10% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity | Pyrococcus furiosus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.002 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409M | Pyrococcus furiosus | |
0.0025 | - |
dATP | 55°C, pH not specified in the publication, wild-type enzyme | Pyrococcus furiosus | |
0.01 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme A408S | Pyrococcus furiosus | |
0.012 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409V | Pyrococcus furiosus | |
0.014 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409I | Pyrococcus furiosus | |
0.046 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410V | Pyrococcus furiosus | |
0.069 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410L | Pyrococcus furiosus | |
0.13 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410I | Pyrococcus furiosus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus furiosus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Pyrococcus furiosus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
dATP + DNAn | - |
Pyrococcus furiosus | diphosphate + DNAn+1 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Pfu Pol | - |
Pyrococcus furiosus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.022 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409I | Pyrococcus furiosus | |
0.028 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409M | Pyrococcus furiosus | |
0.029 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410V | Pyrococcus furiosus | |
0.034 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409V | Pyrococcus furiosus | |
0.037 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme A408S | Pyrococcus furiosus | |
0.22 | - |
dATP | 55°C, pH not specified in the publication, wild-type enzyme | Pyrococcus furiosus | |
0.31 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410L | Pyrococcus furiosus | |
0.32 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410I | Pyrococcus furiosus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410V | Pyrococcus furiosus | |
2 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409I | Pyrococcus furiosus | |
2 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410I | Pyrococcus furiosus | |
3 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409V | Pyrococcus furiosus | |
4 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme A408S | Pyrococcus furiosus | |
4 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme Y410L | Pyrococcus furiosus | |
9 | - |
dATP | 55°C, pH not specified in the publication, wild-type enzyme | Pyrococcus furiosus | |
14 | - |
dATP | 55°C, pH not specified in the publication, mutant enzyme L409M | Pyrococcus furiosus |