Table 3. Kinetic Rate and Binding Constants for WT versus I260Q Nucleotide Incorporation into 19/36AP(T)/15 and 19/36AP(C)/15 DNA Substrates at pH 7.6.
| T:A (matched) |
T:G (mismatched) |
|||
|---|---|---|---|---|
| wild-type | I260Q | wild-type | I260Q | |
| kpol | 13.4 ± 0.5 s−1 | 13.4 ± 0.4 s−1 | 0.26 ± 0.01 s−1 | 1.8 ± 0.03 s−1 |
| Kd,app | 2.2 ± 0.3 μM | 1.1 ± 0.2 μM | 654 ± 42 μM | 88 ± 6 μM |
| kpol/Kd,appa | 6.1 | 12 | 0.00040 | 0.021 |
| fidelityb | 15100 | 590 | ||
| C:G (matched) |
C:A (mismatched) |
|||
|---|---|---|---|---|
| wild-type | I260Q | wild-type | I260Q | |
| kpol | 7.2 ± 0.1 s−1 | 6.6 ± 0.2 s−1 | 0.19 ± 0.01 s−1 | 1.1 ± 0.01 s−1 |
| Kd,app | 1.0 ± 0.1 μM | 0.46 ± 0.07 μM | 298 ± 23 μM | 69 ± 3 μM |
| kpol/Kd,appa | 7.0 | 14 | 0.00064 | 0.016 |
| fidelityb | 10900 | 891 | ||
a
Catalytic efficiency as measured in units of μM−1 s−1.
b
Fidelity defined as [(kpol/Kd,app)cor + (kpol/Kd,app)inc]/(kpol/Kd,app)inc, where the subscripts “cor” and “inc” indicate correct (matched) and incorrect (mismatched) nucleotide incorporation, respectively.