TABLE 1.
Steady-state kinetic parameters for RocR and its mutantsa
| Enzyme | kcat (s−1) | Km (μM) | kcat/Km (s−1 μM−1) | Decrease in kcat (fold) |
|---|---|---|---|---|
| RocR | 0.67 ± 0.03 | 3.2 ± 0.3 | 0.21 | |
| Q161A | 0.13 ± 0.01 | 6.3 ± 1.0 | (2.1 ± 0.4) × 10−2 | 5.1 |
| E175A | NDb | >105 | ||
| R179A | (2.3 ± 0.1) × 10−2 | 8.0 ± 0.9 | (2.9 ± 0.3) × 10−3 | 29.1 |
| N233A | ND | >105 | ||
| E265A | ND | >105 | ||
| T267A | (4.9 ± 0.1) × 10−2 | 2.2 ± 0.4 | (2.2 ± 0.4) × 10−2 | 13.4 |
| E268A | ND | >105 | ||
| E268Q | (1.5 ± 0.1) × 10−3 | 0.3 ± 0.1 | (4.8 ± 0.5) × 10−3 | 446 |
| D295A | ND | >105 | ||
| D296A | (2.1 ± 0.3) × 10−2 | 8.6 ± 2.8 | (2.7 ± 0.9) × 10−3 | 33.5 |
| K316A | ND | >105 | ||
| D318A | (8.0 ± 0.5) × 10−2 | 5.9 ± 1.5 | (1.4 ± 0.4) × 10−2 | 8.4 |
| E352A | ND | >105 | ||
| E352C | ND | >105 | ||
| E352Q | (1.1 ± 0.1) × 10−5 | 3.8 ± 0.7 | (2.9 ± 0.6) × 10−6 | 6.1 × 104 |
| E352D | (2.2 ± 0.1) × 10−5 | 2.2 ± 0.5 | (1 ± 0.2) × 10−5 | 3.0 × 104 |
| E355A | 0.51 ± 0.07 | 2.6 ± 1.1 | 0.19 ± 0.09 | 1.3 |
| Q372A | (8.0 ± 0.5) × 10−2 | 1.3 ± 0.3 | (6.2 ± 1.5) × 10−2 | 8.4 |
a
Conditions were 100 mM Tris buffer (pH 8.0) (23°C), 20 mM KCl, 25 mM MgCl2.
b
ND, not determined due to inactivity or extremely low activity (<105-fold less active than wild-type RocR).