Fang Zheng and Chang-Guo Zhan

Molecular Modelling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, USA. E-mail: fzhen2@email.uky.edu

It has been known that cocaine produces the toxic and physiological effects through not only cocaine itself but also norcocaine formed from cocaine oxidation catalyzed by microsomal cytochrome P450 3A4 in the human liver. In addition, majority of cocaine users also consume alcohol, and alcohol can react with cocaine to produce cocaethylene which is significantly more cytotoxic than cocaine. Hence, a truly valuable cocaine-metabolizing enzyme for cocaine abuse/overdose treatment should be efficient for not only cocaine itself, but also its toxic metabolites including norcocaine and cocaethylene. The catalytic parameters (k_cat and K_M) of human butyrylcholinesterase (BChE) and various mutants (including the one which is under clinical trial for cocaine addiction treatment) for norcocaine and cocaethylene have been characterized in the present study in comparison with those for cocaine. Based on the obtained kinetic data, wild-type human BChE has a lower catalytic activity for norcocaine and cocaethylene compared to its catalytic activity for cocaine. The BChE mutants have considerably improved catalytic activity against norcocaine and cocaethylene compared to the wild-type BChE. One of the BChE mutants, denoted as E12-7, is identified as the most efficient enzyme for hydrolyzing norcocaine and cocaethylene in addition to its high activity for cocaine. E12-7 has a 2020-fold improved catalytic efficiency for cocaine (k_cat = 5700 min^-1, K_M = 3.1 mM, and k_cat/K_M = 1.84 × 10⁹ M^-1 min^-1), a 1080-fold improved catalytic efficiency for norcocaine (k_cat = 2610 min^-1, K_M = 13 mM, and k_cat/K_M = 2.01 × 10⁸ M^-1 min^-1), and an 861-fold improved catalytic efficiency for cocaethylene (k_cat = 3600 min^-1, K_M = 9.5 mM, and k_cat/K_M = 3.79 × 10⁸ M^-1 min^-1). It has been demonstrated that E12-7 as an exogenous enzyme can indeed rapidly metabolize cocaine, norcocaine, and cocaethylene in rats.