Table 2. In vitro DUB activity of structure-guided mutants of TYMV PRO/DUB.
| PRO/DUB proteins | Kapp (% of WT) |
|---|---|
| WT | 100 |
| P866G/P867G | 3.5 ± 0.4 |
| G865A | 32 ± 2 |
| D843A | 28 ± 2 |
| I847A | 22 ± 4 |
| I847D | 1.7 ± 0.1 |
DUB activity of recombinant PRO (wild-type and structure-guided mutants) was measured by a fluorescence assay using Ub-AMC as substrate. Kapp was determined according to the equation V / [E] = Kapp [S], where V is the initial velocity calculated from the kinetic data, and [E] and [S] are the corresponding enzyme and substrate concentrations. To overcome the observed variation in Kapp values according to the batch of Ub-AMC used, values of mutant proteins were expressed as the percentage of that of WT protein measured in the same experiment. Such variations may possibly account for the apparently 3-fold lower activities we previously reported for the I847A and I847D mutants [21].