Table 1.
Steady-state kinetic parameters and relative expression levels for HisA and evolved enzymes
| Enzyme | TrpF kcat, s−1 | TrpF KM, mM | TrpF kcat/KM, s−1 M−1 | HisA kcat, s−1 | HisA KM, μM | HisA kcat/KM, s−1 M−1 | Relative expression |
| HisA | ND | ND | ND | 7.8 ± 2.4 | 17 ± 0.1 | 4.5 × 105 | 1.00 ± 0.01 |
| HisA(dup13-15) | >0.15 | >2 | 75 ± 2 | ND | ND | ND | 0.64 ± 0.02 |
| HisA(dup13-15/D10G) | 0.09 ± 0.02 | 2.1 ± 1.0 | 51 ± 14 | 0.05 ± 0.01 | 1.7 ± 0.2 | 2.8 × 104 | 0.56 ± 0.02 |
| HisA(dup13-15/D10G/G102A) | >0.44 | >2 | 220 ± 30 | 0.05 ± 0.02 | 5.7 ± 1.6 | 9.2 × 103 | 0.52 ± 0.03 |
| HisA(D10G/G102A) | ND | ND | ND | 3.9 ± 0.1 | 24 ± 3 | 1.6 × 105 | 0.67 ± 0.01 |
| HisA(dup13-15/D10G/G102A/Q24L) | >0.52 | >2 | 260 ± 30 | 0.05 ± 0.01 | 10 ± 2 | 5.1 × 103 | 0.36 ± 0.04 |
| HisA(dup13-15/D10G/G102A/Q24L/V106L) | >0.53 | >2 | 260 ± 70 | ND | ND | ND | 0.53 ± 0.01 |
| HisA(dup13-15/D10G/G102A/Q24L/V15[b]M) | >3.6 | >2 | (1.8 ± 0.1) × 103 | ND | ND | ND | 0.36 ± 0.02 |
| HisA(dup13-15/D10G/G102A/Q24L/G44E) | >0.29 | >2 | 140 ± 60 | 0.18 ± 0.02 | 35 ± 2 | 5.2 × 103 | 0.41 ± 0.06 |
| HisA(dup13-15/D10G/G102A/G11D/G44E) | >0.26 | >2 | 130 ± 20 | 0.67 ± 0.05 | 100 ± 20 | 6.7 × 103 | 0.65 ± 0.01 |
| HisA(D10G) | ND | ND | ND | 7.6 ± 0.1 | 29 ± 10 | 2.6 × 105 | 0.77 ± 0.03 |
ND, not detected. SEs for steady-state kinetic parameters are from two independent enzyme preparations, each assayed in triplicate. Except for HisA(dup13-15/D10G), the HisA variants could not be saturated with PRA, so it was only possible to estimate kcat/KM, and then infer kcat. Expression levels are reported relative to HisA, with SEs from two independent experiments.