Table 3. Reactivation of wild type AtSS1 and mutant variants after oxidative inactivation.
Recombinant AtSS1 was either untreated or treated with 0.3 mM dithiothreitol (DTTred) or 5 μM CuCl2. Subsequently, proteins were desalted to remove reducing or oxidizing agents and the reduced (DTTred) protein was incubated with water (2. reduced), while the oxidized (CuCl2) protein was incubated with either water (3. oxidized) or 0.3 mM DTTred (4. reactivated). Enzyme activity was analyzed by SCGA with maltotriose as acceptor. The lower activity for reduced protein samples, as compared to values reported in Table 1, S2 and S4 Tables and Fig 4, is due to the different purification method applied (see details in the Supporting Information, S1 Protocol).
| Protein | Absolute activity, turnovers min-1 | Relative activity | |||
|---|---|---|---|---|---|
| 1. Untreated | 2. Reduced | 3. Oxidized | 4. Reactivated | 5. Reactivation (%) | |
| WT | 53 | 92.7 | 1.70 | 78.6 | 85 |
| C209S | 1.52 | 80.2 | 1.44 | 53.2 | 66 |
| C261S | 1.88 | 79.4 | 1.40 | 55.7 | 70 |
| C442S | 1.45 | 90.2 | 1.18 | 8.53 | 9.5 |
| C458S | 1.45 | 73.5 | 0.77 | 26.0 | 35 |
| C533S | 1.13 | 78.8 | 1.21 | 34.9 | 44 |
| C164S_C545S | 7.06 | 10.4 | 3.66 | 5.09 | 49 |