Table 2.
Stability and Kinetic Parameters of Human and Designed AChE Variants
| AChE Variant | Muta | Normalized Activityb | Inactivation Temperature (°C)c |
ki (SP-VX) × 107 (M−1min−1) | ACh Hydrolysis |
|||
|---|---|---|---|---|---|---|---|---|
| Lysate | Purified | KM (mM) | kcat × 105 (min−1) | kcat/KM × 109 (M−1min−1) | ||||
| hAChE (HEK293) | – | – | – | 50.6 ± 0.3 | 7.92 ± 0.15 | 0.09 ± 0.01 | 3.8 ± 0.2 | 4.4 ± 0.6 |
| hAChE (bacterial) | – | 1 | 44 ± 0.4 | NDd | NDd | NDd | NDd | NDd |
| dAChE1 | 17 | 119 ± 20 | 60.5 ± 0.4 | NDd | 6.48 ± 0.71 | 0.050 ± 0.006 | 4.4 ± 0.1 | 8.7 ± 1.1 |
| dAChE2 | 30 | 280 ± 40 | 61.5 ± 0.5 | 67.1 ± 0.3 | NDd | NDd | NDd | NDd |
| dAChE3 | 42 | 308 ± 44 | 62.3 ± 0.2 | 69.4 ± 0.3 | 2.65 ± 0.52 | 0.177 ± 0.010 | 4.4 ± 0.1 | 2.5 ± 0.2 |
| dAChE4 | 51 | 1770 ± 258 | 62.3 ± 0.3 | 66.2 ± 1.2 | 7.60 ± 0.34 | 0.071 ± 0.007 | 2.73 ± 0.07 | 3.9 ± 0.4 |
| dAChE5 | 67 | 637 ± 134 | 61.1 ± 0.2 | 68.5 ± 0.6 | 6.47 ± 0.82 | 0.104 ± 0.010 | 3.16 ± 0.01 | 3.0 ± 0.3 |
a
Number of amino acid mutations relative to wild-type hAChE.
b
Activity in crude lysates of cells expressing the AChE variants from 250 ml E. coli cultures.
c
The temperature at which 50% of activity was retained. Enzyme samples were incubated for 30 min at varying temperatures and tested for AChE activity after cooling.
d
Not determined.