Table 2.
Summary of kinetic parameters of fully characterized PLpro mutants.
| Mutant | Km (μM) | kcat (min−1) | Fold activity | kcat/Km (μM−1 min−1) | Fold efficiency | Selectivity |
|---|---|---|---|---|---|---|
| ISG15-AMCa | ||||||
| WT | 0.73 ± 0.10 | 10.05 ± 0.44 | 1.0 | 13.81 ± 2.04 | 1.0 | 6.2 |
| R1649A | 2.16 ± 0.33 | 22.84 ± 1.51 | 2.3 | 10.60 ± 1.76 | 0.8 | 0.3 |
| R1649E | 1.50 ± 0.21 | 10.69 ± 0.57 | 1.1 | 7.13 ± 1.06 | 0.5 | 0.2 |
| H1652F | 2.80 ± 0.69 | 22.65 ± 2.63 | 2.3 | 8.08 ± 2.20 | 0.6 | 2.1 |
| T1653R | 2.82 ± 0.74 | 22.73 ± 2.80 | 2.3 | 8.07 ± 2.33 | 0.6 | 12.5 |
| K1657Eb | N.S.c | N.S. | N.S. | 0.74 ± 0.17 | 0.1 | 0.2 |
| V1691F | 0.25 ± 0.04 | 5.20 ± 0.17 | 0.52 | 20.80 ± 3.13 | 1.5 | 4.1 |
| V1691Db | N.S. | N.S. | N.S. | 0.23 ± 0.05 | 0.017 | 0.1 |
| V1691S | N.S. | N.S. | N.S. | 2.23 ± 0.10 | 0.2 | 1.6 |
| Ub-AMCa | ||||||
| WT | 16.58 ± 4.20 | 37.07 ± 6.21 | 1.0 | 2.24 ± 0.68 | 1.0 | 0.2 |
| R1649A | 1.13 ± 0.20 | 46.51 ± 2.36 | 1.3 | 41.31 ± 7.58 | 18.5 | 3.9 |
| R1649E | 1.65 ± 0.21 | 62.62 ± 2.57 | 1.7 | 37.93 ± 5.08 | 17.0 | 5.3 |
| H1652F | 1.77 ± 0.21 | 6.76 ± 0.26 | 0.2 | 3.81 ± 0.47 | 1.7 | 0.5 |
| T1653R | N.S. | N.S. | N.S. | 0.55 ± 0.01 | 0.2 | 0.1 |
| K1657Eb | 3.17 ± 0.37 | 12.22 ± 0.43 | 0.3 | 3.85 ± 0.12 | 1.7 | 5.2 |
| V1691F | 26.17 ± 3.01 | 132.30 ± 11.29 | 3.6 | 5.06 ± 0.72 | 2.3 | 0.2 |
| V1691Db | 10.04 ± 2.33 | 29.44 ± 2.40 | 0.8 | 2.93 ± 0.25 | 1.3 | 12.7 |
| V1691S | N.S. | N.S. | N.S. | 1.37 ± 0.02 | 0.6 | 0.6 |
a
Steady-state values were determined from duplicate measurements, reported as a mean ± standard error.
b
Kinetic parameters from Daczkowski, C.M. et al. 2017b.
c
NS, enzyme not saturated. kapp values reported as a slope ± best-fit error from a linear regression were used to approximate kcat/Km.