Table 4.
Steady-state kinetic parameters for hpol κ19-526 catalysis on G4 DNA and non-G4 DNA substrates prepared in LiCl
| kcat (min−1) | KM,dNTP (μM) | kcat/KM,dNTP (min−1 μM−1) | Relative activitya | |
|---|---|---|---|---|
| 13/42-mer: dCMP insertion opposite template dG −10 nt from G22 | ||||
| non-G4 DNA | 8.8 ± 0.4 | 18 ± 3 | 0.49 | - |
| G4 DNA | 9.0 ± 0.8 | 17 ± 4 | 0.53 | 108% |
| 14/42-mer: dGMP insertion opposite template dC −9 nt from G22 | ||||
| non-G4 DNA | 1.6 ± 0.1 | 42 ± 7 | 0.039 | - |
| G4 DNA | 1.6 ± 0.2 | 47 ± 20 | 0.034 | 87% |
| 18/42-mer: dAMP insertion opposite template dT −5 nt from G22 | ||||
| non-G4 DNA | 6.8 ± 0.3 | 4.9 ± 0.8 | 1.39 | - |
| G4 DNA | 6.3 ± 0.3 | 7.5 ± 1 | 0.84 | 60% |
| 19/42-mer: dCMP insertion opposite template dG −4 nt from G22 | ||||
| non-G4 DNA | 9.3 ± 0.6 | 26 ± 5 | 0.36 | - |
| G4 DNA | 7.6 ± 0.7 | 25 ± 6 | 0.30 | 83% |
| 20/42-mer: dGMP insertion opposite template dC −3 nt from G22 | ||||
| non-G4 DNA | 1.4 ± 0.04 | 3.7 ± 0.4 | 0.38 | - |
| G4 DNA | 2.4 ± 0.07 | 4.8 ± 0.6 | 0.50 | 132% |
| 21/42-mer: dCMP insertion opposite template dG −2 nt from G22 | ||||
| non-G4 DNA | 3.8 ± 0.2 | 31 ± 4 | 0.12 | - |
| G4 DNA | 2.6 ± 0.2 | 23 ± 6 | 0.11 | 92% |
| 22/42-mer: dAMP insertion opposite template dT −1 nt from G22 | ||||
| non-G4 DNA | 1.2 ± 0.03 | 3.4 ± 0.4 | 0.35 | - |
| G4 DNA | 1.1 ± 0.03 | 3.9 ± 0.5 | 0.28 | 80% |
| 23/42-mer: dCMP insertion opposite G22 | ||||
| non-G4 DNA | 1.2 ± 0.05 | 19 ± 2 | 0.063 | - |
| G4 DNA | 1.0 ± 0.04 | 28 ± 3 | 0.036 | 57% |
a
Relative activity was calculated as [(kcat/Km,dNTP)G4 DNA/(kcat/Km,dNTP)non-G4 DNA] × 100. For determination of kinetic values, graphs of the initial rate of product formation versus dNTP concentration were plotted using non-linear regression analysis (one-site hyperbolic fit) in the GraphPad Prism program. The standard error of the fit is reported for each value.