Table 1.
Kinetic characterization of mutant sortases
| kcat, s-1 | Km LPETG, mM | kcat/Km LPETG, M-1 s-1 | Km GGG-COOH, μM | |
| WT | 1.5 ± 0.2 | 7.6 ± 0.5 | 200 ± 30 | 140 ± 30 |
| D160N/K190E/K196T (clone 4.2) | 3.7 ± 0.6 | 1.6 ± 0.4 | 2,400 ± 700 | 1,200 ± 200 |
| P94S/D165A (clone 4.3) | 2.9 ± 0.0 | 1.1 ± 0.1 | 2,600 ± 100 | 1,700 ± 400 |
| P94S/D160N/D165A/K196T | 4.8 ± 0.8 | 0.17 ± 0.03 | 28,000 ± 7,000 | 4,800 ± 700 |
| P94S/D160N/K196T | 4.8 ± 0.6 | 0.56 ± 0.07 | 8,600 ± 1,500 | 1,830 ± 330 |
| P94S/D160N/D165A | 3.8 ± 0.2 | 0.51 ± 0.38 | 7,500 ± 300 | 1,750 ± 250 |
| P94R/D160N/D165A/K190E/K196T | 5.4 ± 0.4 | 0.23 ± 0.02 | 23,000 ± 3,000 | 2,900 ± 200 |
| P94S | 1.6 ± 0.1 | 2.5 ± 0.6 | 600 ± 200 | 650 ± 150 |
| D160N | 2.3 ± 0.2 | 3.7 ± 0.5 | 600 ± 100 | 330 ± 20 |
| D165A | 2.4 ± 0.3 | 3.6 ± 1.0 | 700 ± 200 | 1,000 ± 480 |
| K196T | 1.2 ± 0.1 | 3.3 ± 0.8 | 400 ± 100 | 200 ± 70 |
Kinetic parameters kcat and Km were obtained from fitting initial reaction rates at 22.5 °C to the Michaelis–Menten equation. Errors represent the standard deviation of three independent experiments.