Table 2.
Experimental kinetic data in comparison with the FEP-calculated cumulative free energy shifts (in kcal/mol) of the free energy change from the free enzyme to the rate-determining transition state for (−)-cocaine hydrolysis catalyzed by the BChE mutants.
| Mutant | Experiment kinetic data | Calc. | ||||
|---|---|---|---|---|---|---|
| kcat (min−1) | KM (μM) | kcat/KM (min−1M−1) | Relative kcat/KMf | ΔΔGg | ΔΔGh | |
| Wild-type BChEa | 4.1 | 4.5 | 9.1 × 105 | 1 | N.A.i | N.A.i |
| A199S/A328W/Y332Gb | 389 | 5.4 | 7.2 × 107 | 79 | 0.00 | 0.00 |
| A199S/F227A/A328W/Y332Gc | 1150 | 3.9 | 3.0 × 108 | 320 | −0.83 | −1.13 |
| A199S/S287G/A328W/Y332Gd | 3060 | 3.1 | 9.9 × 108 | 1080 | −1.55 | −0.95 |
| A199S/F227A/S287A/A328W/Y332Ge | 5700 | 3.1 | 1.8 × 109 | 2020 | −1.92 | −2.00 |
The experimental data from ref. 11
The experimental data from ref. 22
The kinetic parameters determined in this study
The experimental data from ref. 24
The experimental data from ref. 23
The relative kcat/KM refers to the ratio of the catalytic efficiency of the BChE mutant to that of the wild-type against (−)-cocaine.
The experimentally-derived cumulative ΔΔG from the A199S/A328W/Y332G mutant to the mutant under consideration.
The computationally determined cumulative ΔΔG from the A199S/A328W/Y332G mutant to the mutant under consideration.
N.A., Not applicable.