Table 2.
Representative kinetic parameters and specific activities of PagF variants using Tyr3 tripeptide as a substrate
| Mutant | [S], mM | Vmax, min−1* | Km, mM | V/K, min−1 mM−1 | Activity, min−1† |
| WT | 1 | 5.7 ± 0.1 | 0.15 ± 0.01 | 38.0 ± 1.0 | 4.99 ± 0.01 |
| H237L | 1 | 17.6 ± 0.4 | 0.32 ± 0.02 | 55.0 ± 2.2 | — |
| F222V | 1 | 8.15 ± 2.05 | 0.68 ± 0.22 | 12.45 ± 0.85 | — |
| H138A | 10 | — | — | — | 3.70 ± 0.09 |
| R65A | 10 | — | — | — | 1.15 ± 0.07 |
| Y235A | 20 | — | — | — | 0.59 ± 0.02 |
| Y290A | 10 | — | — | — | 0.26 ± 0.01 |
Measurements were conducted in duplicate. SI Appendix, Fig. S12 presents progress curves used to calculate the parameters and the activities.
*
The maximum rate given by the fit depends on the concentration of enzyme in the assay. Vmax was calculated from this maximum rate and the enzyme concentration.
†
Turnovers per min by a single enzyme molecule. Initial rates were calculated from the linear part of each assay.