Table 3.
Properties of wtPTE InDel variants selected for improved arylesterase activity.
| AE | PTE | Thermal denaturation | |||||
|---|---|---|---|---|---|---|---|
| PTE varianta | kcat (s−1) | KM (µM) | kcat/KM (M−1 s−1) | kcat (s−1) | KM (µM) | kcat/KM (M−1 s−1) | Tm (°C)d |
| wtPTEb | 0.075 ± 0.004 | 179 ± 21 | (4.2 ± 0.6) × 102 | 1270 ± 27 | 57 ± 5 | (2.2 ± 0.2) × 107 | 78.1 ± 0.2 |
| H254Rb | 0.27 ± 0.01 | 250 ± 32 | (1.1 ± 0.2) × 103 | 62 ± 3 | 7 ± 1 | (8.9 ± 1.4) × 106 | 88 ± 0.1 |
| ∆A270L271L272G273c | 2.1 ± 0.1 | 258 ± 40 | (8.2 ± 1.4) × 103 | 55 ± 4 | 148 ± 22 | (3.7 ± 0.7) × 105 | 82 ± 1 |
| P256R/G256aA256bc | 4.6 ± 0.3 | 381 ± 59 | (1.2 ± 0.3) × 104 | 54 ± 3 | 988 ± 114 | (5.4 ± 0.7) × 104 | 84.3 ± 0.3 |
| S256aG256bc | 8.9 ± 0.7 | 821 ± 152 | (1.1 ± 0.3) × 104 | 13 ± 1 | 116 ± 21 | (1.1 ± 0.3) × 105 | 77.5 ± 0.4 |
| G311ac | 4.2 ± 0.2 | 292 ± 34 | (1.5 ± 0.2) × 104 | 39 ± 1 | 307 ± 25 | (1.3 ± 0.2) × 105 | 75.2 ± 0.3 |
AE, arylesterase (substrate: 2-NH); PTE, phosphotriesterase (substrate: paraoxon).
aThe symbol ∆ before a residue (or a group of residues) signifies that this (or these) residue(s) have been deleted. Inserted residues are labelled using the number of the position after which they are inserted and alphabetical order (e.g., glutamine and tyrosine residues inserted in this order after the residues at position 230 would be labelled Q230aY230b).
bKinetic parameters are from Tokuriki et al.18.
cSee Supplementary Fig. S15 for detailed experimental conditions for Michaelis–Menten kinetics.
dThermal denaturation for wtPTE and all InDel variants was measured with SYPRO Orange as the fluorescent probe and Tm is given as mean ± standard deviation (from six or more measurements; Supplementary Fig. S16). For variant H254R, the Tm value is from Wyganowki et al.61.