TABLE 3.
Transient state kinetic constants for reduction of VPI of native enzyme and directed variants by VA and RB5
Shown are first-order rate constants (k2), equilibrium dissociation constants (Kd2) and apparent second-order rate constants (k2app). Hill equation used for RB5 k2 and Kd2 estimation due to sigmoidal kinetic curves (supplemental Fig. 4B). Means and 95% confidence limits.
| k2 | Kd2 (0.5) | k2app | |
|---|---|---|---|
| s−1 | m | m−1 · s−1 | |
| VPI reduction by VA | |||
| VP | 168 ± 13 | (60 ± 7) × 10−3 | (2.8 ± 0.1) × 103 |
| W164Y | 36 ± 5 | (114 ± 20) × 10−3 | (3.2 ± 0.1) × 102 |
| W164H | -a | - | (2.9 ± 0.1) × 102 |
| W164S | - | - | (5.6 ± 0.6) × 101 |
| R257A/A260F | - | - | (6.2 ± 0.1) × 104 |
| W164Y/R257A/A260F | - | - | (2.3 ± 0.1) × 102 |
| VPI reduction by RB5 | |||
| VP | - | - | (3.9 ± 0.1) × 106 |
| W164Y | 5.2 ± 0.1 | (21.3 ± 0.7) × 10−6 | 2.5 × 105 |
| W164H | 21.5 ± 2.2 | (41.5 ± 5.1) × 10−6 | 5.2 × 105 |
| W164S | 6.8 ± 1.2 | (29.1 ± 0.7) × 10−6 | 2.3 × 105 |
| R257A/A260F | - | - | ∼3.0 × 107b |
| W164Y/R257A/A260F | 23.7 ± 2.7 | (29.4 ± 3.5) × 10−6 | 8.1 × 105 |
a
-, Not determined because of non-saturation kinetics.
b
Measured at 10 °C.