TABLE 2.
Kinetic parameters of wild-type, recombinant, and evolved UPO variants
| Substrate | Kinetic constant | Valuea |
||
|---|---|---|---|---|
| wtUPO1 | n*-UPO1 | PaDa-I | ||
| ABTS | Km (mM) | 0.025 ± 0.002 | 0.027 ± 0.005 | 0.048 ± 0.004 |
| kcat (s−1) | 221 ± 6 | 45.0 ± 2.7 | 395 ± 13 | |
| kcat/Km (mM−1 s−1) | 8,800 ± 692 | 1,600 ± 37 | 8,200 ± 598 | |
| NBD | Km (mM) | 0.684 ± 0.207 | 0.782 ± 0.352 | 0.483 ± 0.095 |
| kcat (s−1) | 219 ± 25 | 31.7 ± 6.1 | 338 ± 22 | |
| kcat/Km (mM−1 s−1) | 320 ± 64 | 38.0 ± 11 | 700 ± 99 | |
| Benzyl alcohol | Km (mM) | 1.90 ± 0.11 | 1.10 ± 0.23 | 2.47 ± 0.32 |
| kcat (s−1) | 329 ± 7 | 44.8 ± 3.1 | 307 ± 15 | |
| kcat/Km (mM−1 s−1) | 174 ± 7 | 41.0 ± 6.3 | 124 ± 11 | |
| Veratryl alcohol | Km (mM) | 5.20 ± 0.31 | 5.30 ± 0.82 | 7.9 ± 0.7 |
| kcat (s−1) | 88 ± 2 | 15.2 ± 1.1 | 121 ± 5 | |
| kcat/Km (mM−1 s−1) | 17 ± 0.7 | 2.9 ± 0.25 | 15 ± 0.9 | |
| H2O2 | Km (mM) | 1.37 ± 0.16 | 0.69 ± 0.20 | 0.49 ± 0.06 |
| kcat (s−1) | 290 ± 15 | 40.9 ± 3.8 | 238 ± 8 | |
| kcat/Km (mM−1 s−1) | 211 ± 15 | 59.0 ± 12.3 | 500 ± 42 | |
ABTS kinetic constants for UPO1 were estimated in 100 mM sodium citrate/phosphate buffer, pH 4.4, containing 2 mM H2O2 and those for the rest of the substrates in 100 mM potassium phosphate buffer, pH 7.0, containing 2 mM H2O2 (benzyl and veratryl alcohols) or 1 mM H2O2 (NBD). H2O2 kinetic constants were estimated using benzyl alcohol as a reducing substrate under the corresponding saturated conditions. wtUPO1, UPO1 wild-type expressed in A. aegerita; n*-UPO, native UPO1 fused to the evolved signal peptide for secretion in S. cerevisiae; PaDa-I mutant, the ultimate variant of the whole evolution process in S. cerevisiae (containing the evolved signal peptide plus the evolved UPO1).