Table 2.
Kinetic and Inhibition Parameters for SARS PLpro and Its Mutants on -AMC Substrates
| SARS PLpro Mutant | Kinetic Parameter | MonoUb-AMC | Triazole Linked DiUbLys48-AMC | Native DiUbLys48-AMC | ISG15-AMC |
|---|---|---|---|---|---|
| WT | Apparent kcat/KM [M−1s−1] | 3.33E+04 | 1.26E+06 | 1.01E+06a | 5.98E+05 |
| kcat [s−1] | 0.5042 ± 0.02839 | 42.02 ± 3.872 | n/a | 9.533 ± 1.218 | |
| KM [μM] | 15.12 ± 1.747 | 33.42 ± 4.869 | n/a | 15.94 ± 3.172 | |
| Fold kcat/KM over monoUb-AMC | 1.00 | 37.70 | 30.33a | 17.93 | |
| Michaelis-Menten curve fit (R2) | 0.9845 | 0.9668 | n/a | 0.9411 | |
| F70S E71K H74G (S2 mutant) | kcat/KM [M−1s−1] | — | 4.23E+04a | — | 2.94E+05 |
| kcat [s−1] | — | n/a | — | 2.748 ± 0.6693 | |
| KM [μM] | — | n/a | — | 9.359 ± 3.547 | |
| % kcat/KM of WT (per substrate) | — | 3.37 | — | 49.10 | |
| R167S E168R (S1 polar mutant) | kcat/KM [M−1s−1] | — | 6.50E+05 | — | 3.64E+04 |
| kcat [s−1] | — | 65.56 ± 22.38 | — | 0.318 ± 0.1184 | |
| KM [μM] | — | 100.8 ± 42.49 | — | 8.764 ± 5.614 | |
| % kcat/KM of WT (per substrate) | — | 51.63 | — | 6.08 | |
| M209S (S1 hydrophobic mutant) | kcat/KM [M−1s−1] | — | 7.06E+05 | — | 4.20E+05 |
| kcat [s−1] | — | 46.59 ± 10.29 | — | 4.774 ± 1.263 | |
| KM [μM] | — | 66.01 ± 19.61 | — | 11.88 ± 4.578 | |
| % kcat/KM of WT (per substrate) | — | 56.13 | — | 67.19 | |
| WT | Ki [μM] with monoUbb | NI | NI | NI | NI |
| Ki [μM] with diUbLys48 | 2.26 (0.9265) | 9.05 (0.8942) | 12.07 (0.8892) | 3.31 (0.8351) | |
| Ki [μM] with triUbLys48 | — | 10.57 (0.9509) | 10.11 (0.9066) | 4.08 (0.6709) | |
| Ki [μM] with ISG15 | NI | NI | NI | NI |
n/a, not applicable (kcat and KM cannot be independently calculated); NI, no detectable inhibition (IC50 > 100 μM or data do not converge).
a
Substrate not saturated, kcat/KM calculated from slope of linear graph.
b
Ki values were derived from IC50 values based on the equation Ki = IC50/(S/KM+1), assuming competitive inhibition, where S is the concentration of the substrate (based on Cer et al., 2009). Brackets show goodness of fit (R2) of IC50 values obtained from Prism’s log(inhibitor) versus normalized curve fit. Inhibition curves are shown in Figure S6A.