Characterization of a high-activity mutant of human butyrylcholinesterase against (−)-cocaine

Abstract

Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant ( k cat) and Michaelis–Menten constant ( K M) for (−)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060 min −1 and 3.1 μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency ( k cat/ K M) against (−)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (−)-cocaine.