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Literature summary extracted from
- Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii (2004), J. Biol. Chem., 279, 16687-16696.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 3.1.99.B1 | expression in Escherichia coli | Pyrococcus horikoshii |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 3.1.99.B1 | F278A/F279A | the kcat values of the mutant enzyme are decreased about 1020% with the 5'-recess-end substrate and the nick substrate | Pyrococcus horikoshii |
| 3.1.99.B1 | F278H/F279H | kcat value decreases 83fold for the 5'-recess-end substrate and 150fold for the nick substrate compared with the wild-type values. 454fold decrease in kcat/Km for the 5'-recess-end substrate, 80fold decrease in kcat/Km for the nick substrate. The Km-value for the endo activity with the double flap substrate is increased 4fold, the kcat/Km-value is decreased 14fold, compared with the wild-type value | Pyrococcus horikoshii |
| 3.1.99.B1 | F278H/F279H | the Km value for the 5'-recess-end substrate is elevated 5 times compared with the wild-type value whereas for the nick substrate the Km value of F278H/F279H is 60% the wild-type value | Pyrococcus horikoshii |
| 3.1.99.B1 | F278L/F279L | Km-values are lower than the wild-type values | Pyrococcus horikoshii |
| 3.1.99.B1 | F278L/F279L | the kcat values of the mutant enzyme are decreased about 1020% with the 5'-recess-end substrate and the nick substrate | Pyrococcus horikoshii |
| 3.1.99.B1 | F278W/F279W | Km-values are lower than the wild-type values | Pyrococcus horikoshii |
| 3.1.99.B1 | F278W/F279W | the kcat values of the mutant enzyme are decreased about 1020% with the 5'-recess-end substrate and the nick substrate | Pyrococcus horikoshii |
| 3.1.99.B1 | F278Y/F279Y | Km-values are lower than the wild-type values | Pyrococcus horikoshii |
| 3.1.99.B1 | F278Y/F279Y | the kcat value of F278Y/F279Y is restored to around 70% of that of wil-type enzyme with the 5'-recess-end substrate and the nick substrate | Pyrococcus horikoshii |
| 3.1.99.B1 | F35A | kcat and Km of F35A and F35L show no significant decrease compared with the wild-type values | Pyrococcus horikoshii |
| 3.1.99.B1 | F35L | kcat and Km of F35A and F35L show no significant decrease compared with the wild-type values | Pyrococcus horikoshii |
| 3.1.99.B1 | F35Y | the Km values of the mutant enzyme are about 4- and 3fold higher than the values of wild-type enzyme with the nick and 5'-recess-end substrates, respectively | Pyrococcus horikoshii |
| 3.1.99.B1 | F79A | for the 5'-recess-end substrate, the kcat value of the mutant enzyme decreases 71fold compared with that of wild-type.For the nick substrate, the kcat value decreases 25fold compared with that of wild-type. 58fold decrease in kcat/Km for the 5'-recess-end substrate, 75fold decrease in kcat/Km for the nick substrate. The Km-value for the endo activity with the double flap substrate is increased 7fold, the kcat/Km-value is decreased 6fold, compared with the wild-type value.For the single flap and pseudo-Y substrates, the kcat of F79A decreases 31- and 37fold, respectively, compared with that of wild-type | Pyrococcus horikoshii |
| 3.1.99.B1 | F79H | te kcat value of the mutant for the 5'-recess-end substrate is decreased to about 50% of the wild-type value, and the kcat value of F79H for the nick substrate is seven times lower than that of wild-type enzyme. The Km value of F79H for the 5'-recess-end substrate is 13fold higher than that of wild-type, whereas the Km value of F79H for the nick substrate is about 2 times higher than that of wild-type | Pyrococcus horikoshii |
| 3.1.99.B1 | F79L | for the 5'-recess-end substrate, the kcat value of the mutant enzyme is restored to 20% of the wild-type value.For the nick substrate, the kcat value of the mutant enzyme is restored to 20% of that of the wild-type enzyme. The kcat-values for of the single flap and pseudo-Y substrates decrease to 17 and 7% of the wild-type values, respectively. The Km for the single flap and pseudo-Y substrates is varied moderately, but not significantly, compared with that of wild-type enzyme | Pyrococcus horikoshii |
| 3.1.99.B1 | F79W | the kcat values of the mutant enzyme for the 5'-recess-end substrate and the nick substrate are restored to almost the same level as the wild-type values | Pyrococcus horikoshii |
| 3.1.99.B1 | F79Y | the kcat values of the mutant enzyme for the 5'-recess-end substrate and the nick substrate are restored to almost the same level as the wild-type values. The kcat-value for the single flap and pseudo-Y substrates are restored to almost the same level as the wild-type substrates | Pyrococcus horikoshii |
| 3.1.99.B1 | Y33A | kcat decreases 333fold, compared with that of the wild-type enzyme, for exo-activity against the 5'-recess-end substrate. For the exo-activity against the nick substrate, the kcat values decreases 53fold.The kcat of Y33A for the single flap substrate decreases 30fold. The kcat of Y33A for the pseudo-Y substrate decreases 485fold | Pyrococcus horikoshii |
| 3.1.99.B1 | Y33F | kcat for exo-activity against the 5'-recess-end substrate is 20-30% of the wild-type value. The kcat values of Y33F for the single flap and pseudo-Y substrates are restored to 38 and 20% of the value of wild-type, respectively | Pyrococcus horikoshii |
| 3.1.99.B1 | Y33H | kcat for exo-activity against the 5'-recess-end substrate is 20-30% of the wild-type value | Pyrococcus horikoshii |
| 3.1.99.B1 | Y33L | kcat decreases 1180fold, compared with that of the wild-type enzyme, for exo-activity against the 5'-recess-end substrate. For the exo-activity against the nick substrate, the kcat values decreases 134fold. 2353fold decrease in kcat/Km for the 5'-recess-end substrate, 270fold decrease in kcat/Km for the nick substrate. The Km-value for the endo activity with the double flap substrate is increased 4fold, the kcat/Km-value is decreased 5fold, compared with the wild-type value. The kcat of Y33A for the single flap substrate decreases 433fold. The kcat of Y33A for the pseudo-Y substrate decreases 3233fold | Pyrococcus horikoshii |
| 3.1.99.B1 | Y33W | kcat for exo-activity against the 5'-recess-end substrate is 20-30% of the wild-type value | Pyrococcus horikoshii |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 3.1.99.B1 | Pyrococcus horikoshii | O50123 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 3.1.99.B1 | - |
Pyrococcus horikoshii |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 3.1.99.B1 | additional information | the aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contribute to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but play minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection) | Pyrococcus horikoshii | ? | - |
? |  |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 3.1.99.B1 | FEN-1 | - |
Pyrococcus horikoshii |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 3.1.99.B1 | 37 | - |
assay at | Pyrococcus horikoshii |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 3.1.99.B1 | 7.4 | - |
assay at | Pyrococcus horikoshii |
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Release 2023.1 (January 2023)
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