TABLE 1.
Catalytic constants for ATCh hydrolysis by wild type and mutant human AChE in 0.1 m phosphate buffer, pH 7.4, at 22 °C
For all studied enzymes, inhibition by excess ATCh was evident except for F297I/Y337A and Y337A/F338A, where ATCh hydrolysis followed simple Michaelis kinetics (b = 1). Hydrolysis by F295L/Y337A/F338A was activated by excess substrate (b >1). kcat values for WT hAChE and mutants F338A and Y337A/F338A were determined as (1.4 ± 0.40)·105 min−1, (0.74 ± 0.21)·105 min−1, and (0.53 ± 0.09)·105 min−1, respectively.
| hAChE | Km | Kss | b |
|---|---|---|---|
| (μm) | (mm) | ||
| WT | 160 ± 12 | 10 ± 1 | 0.097 ± 0.019 |
| E202Q | 370 ± 20 | 64 ± 19 | 0.24 ± 0.08 |
| F295L | 530 ± 54 | 43 ± 13 | 0.17 ± 0.08 |
| F297I | 2800 ± 170 | 240 ± 25 | 0 |
| Y337A | 130 ± 10 | 6.9 ± 1.5 | 0.54 ± 0.02 |
| F338A | 180 ± 23 | 3.0 ± 0.8 | 0.42 ± 0.03 |
| F295L/Y337A | 710 ± 31 | 420 ± 45 | 0 |
| F297I/Y337A | 4000 ± 140 | 1.0 | |
| E202Q/F338A | 670 ± 300 | 4.1 ± 3.8 | 0.46 ± 0.13 |
| E202Q/Y337A | 200 ± 20 | 7.7 ± 2.5 | 0.53 ± 0.03 |
| F295L/F338A | 350 ± 18 | 110 ± 7 | 0 |
| Y337A/F338A | 270 ± 18 | 1.0 | |
| D134H/Y337A | 270 ± 27 | 7.5 ± 2.1 | 0.51 ± 0.02 |
| D134H/F338A | 310 ± 21 | 6.0 ± 1.1 | 0.52 ± 0.02 |
| E202Q/F295L/Y337A | 360 ± 26 | 25 ± 6 | 0.39 ± 0.03 |
| E202Q/F297I/Y337A | 610 ± 38 | 660 ± 140 | 0 |
| F295L/Y337A/F338A | 1700 ± 250 | 44 ± 8 | 3.00 ± 0.21 |