Kinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene

It is known that the majority of cocaine users also consume alcohol. Alcohol can react with cocaine to produce a significantly more cytotoxic compound, cocaethylene. Hence a truly valuable cocaine-metabolizing enzyme as treatment for cocaine abuse/overdose should be efficient for not only cocaine itself, but also cocaethylene. The catalytic parameters (k_cat and K_M) of human BChE (butyrylcholinesterase) and two mutants (known as cocaine hydrolases E14-3 and E12-7) for cocaethylene are characterized in the present study, for the first time, in comparison with those for cocaine. On the basis of the obtained kinetic data, wild-type human BChE has a lower catalytic activity for cocaethylene (k_cat=3.3 min⁻¹, K_M=7.5 μM and k_cat/K_M=4.40×10⁵ M⁻¹·min⁻¹) compared with its catalytic activity for (−)-cocaine. E14-3 and E12-7 have a considerably improved catalytic activity against cocaethylene compared with the wild-type BChE. E12-7 is identified as the most efficient enzyme for hydrolysing cocaethylene in addition to its high activity for (−)-cocaine. E12-7 has an 861-fold improved catalytic efficiency for cocaethylene (k_cat=3600 min⁻¹, K_M=9.5 μM 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 cocaethylene in rats. Further kinetic modelling has suggested that E12-7 with an identical concentration as that of the endogenous BChE in human plasma can effectively eliminate (−)-cocaine, cocaethylene and norcocaine in simplified kinetic models of cocaine abuse and overdose associated with the concurrent use of cocaine and alcohol.