Table 1.
Kinetic parameters determined in vitro for (−)-cocaine, norcocaine and cocaethylene hydrolyses catalyzed by wild-type BChE and its mutants.
Substrate | Enzymea | KM (μM) | kcat (min−1) | kcat/KM (M−1min−1) | RCEd |
---|---|---|---|---|---|
(−)-Cocaineb | WT BChE | 4.5 | 4.1 | 9.11 × 105 | 1 |
E14-3 | 3.1 | 3,060 | 9.87 × 108 | 1,080 | |
E12-7 | 3.1 | 5,700 | 1.84 × 109 | 2,020 | |
Norcocaineb | WT BChE | 15 | 2.8 | 1.87 × 105 | 1 |
E14-3 | 12 | 766 | 6.38 × 107 | 343 | |
E12-7 | 13 | 2,610 | 2.01 × 108 | 1,080 | |
Cocaethylenec | WT BChE | 7.5 | 3.3 | 4.40 × 105 | 1 |
E14-3 | 8.0 | 1,820 | 2.28 × 108 | 517 | |
E12-7 | 9.5 | 3,600 | 3.79 × 108 | 861 |
The enzyme under the study was wild-type human BChE (WT BChE), A199S/S287G/A328W/Y332G mutant (E14-3), (CocH2), or A199S/F227A/S287G/A328W/Y332G mutant (E12-7).
Data for wild-type BChE against (−)-cocaine came from reference [12], data for E14-3 against (−)-cocaine came from reference [25], data for CocH3 against (−)-cocaine came from reference [22], and data for all enzymes against norcocaine came from reference [[26]].
All of the kinetic data for cocaethylene were determined in the present study for the first time.
RCE refers to the relative catalytic efficiency (kcat/KM), i.e. the ratio of the kcat/KM value of the mutant to that of wild-type BChE against the same substrate.