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Literature summary extracted from

  • Kennedy, E.M.; Hergott, C.; Dewhurst, S.; Kim, B.
    The mechanistic architecture of thermostable Pyrococcus furiosus family B DNA polymerase motif A and its interaction with the dNTP substrate (2009), Biochemistry, 48, 11161-11168.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.7 overexpression in Escherichia coli from pET28a plasmids Pyrococcus furiosus

Protein Variants

EC Number Protein Variants Comment Organism
2.7.7.7 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
2.7.7.7 D215A 3'-5'-exonuclease inactive mutant enzyme Pyrococcus furiosus
2.7.7.7 L409I kcat/Km for dATP is about 20% compared to the wild-type enzyme Pyrococcus furiosus
2.7.7.7 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
2.7.7.7 L409V kcat/Km for dATP is about 35% compared to the wild-type enzyme Pyrococcus furiosus
2.7.7.7 Y410I kcat/Km for dATP is about 20% compared to the wild-type enzyme Pyrococcus furiosus
2.7.7.7 Y410L kcat/Km for dATP is about 45% compared to the wild-type enzyme Pyrococcus furiosus
2.7.7.7 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.7.7 0.002
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409M Pyrococcus furiosus
2.7.7.7 0.0025
-
 dATP 55°C, pH not specified in the publication, wild-type enzyme Pyrococcus furiosus
2.7.7.7 0.01
-
 dATP 55°C, pH not specified in the publication, mutant enzyme A408S Pyrococcus furiosus
2.7.7.7 0.012
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409V Pyrococcus furiosus
2.7.7.7 0.014
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409I Pyrococcus furiosus
2.7.7.7 0.046
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410V Pyrococcus furiosus
2.7.7.7 0.069
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410L Pyrococcus furiosus
2.7.7.7 0.13
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410I Pyrococcus furiosus

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.7 Pyrococcus furiosus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.7
-
 Pyrococcus furiosus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.7 dATP + DNAn
-
 Pyrococcus furiosus diphosphate + DNAn+1
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.7.7.7 Pfu Pol
-
 Pyrococcus furiosus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.7.7 0.022
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409I Pyrococcus furiosus
2.7.7.7 0.028
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409M Pyrococcus furiosus
2.7.7.7 0.029
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410V Pyrococcus furiosus
2.7.7.7 0.034
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409V Pyrococcus furiosus
2.7.7.7 0.037
-
 dATP 55°C, pH not specified in the publication, mutant enzyme A408S Pyrococcus furiosus
2.7.7.7 0.22
-
 dATP 55°C, pH not specified in the publication, wild-type enzyme Pyrococcus furiosus
2.7.7.7 0.31
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410L Pyrococcus furiosus
2.7.7.7 0.32
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410I Pyrococcus furiosus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.7.7.7 1
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410V Pyrococcus furiosus
2.7.7.7 2
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409I Pyrococcus furiosus
2.7.7.7 2
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410I Pyrococcus furiosus
2.7.7.7 3
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409V Pyrococcus furiosus
2.7.7.7 4
-
 dATP 55°C, pH not specified in the publication, mutant enzyme A408S Pyrococcus furiosus
2.7.7.7 4
-
 dATP 55°C, pH not specified in the publication, mutant enzyme Y410L Pyrococcus furiosus
2.7.7.7 9
-
 dATP 55°C, pH not specified in the publication, wild-type enzyme Pyrococcus furiosus
2.7.7.7 14
-
 dATP 55°C, pH not specified in the publication, mutant enzyme L409M Pyrococcus furiosus