A majority of
cocaine users also consume alcohol. The concurrent use of
cocaine and alcohol produces the pharmacologically active metabolites
cocaethylene and
norcocaethylene, in addition to
norcocaine. Both
cocaethylene and
norcocaethylene are more toxic than
cocaine itself. Hence, a truly valuable
cocaine-metabolizing
enzyme for
cocaine abuse/overdose treatment should be effective for the hydrolysis of not only
cocaine, but also its metabolites
norcocaine,
cocaethylene, and
norcocaethylene. However, there has been no report on
enzymes capable of hydrolyzing
norcocaethylene (the most toxic metabolite of
cocaine). The catalytic efficiency parameters (kcat and KM) of human
butyrylcholinesterase (BChE) and two mutants (known as
cocaine hydrolases E14-3 and E12-7) against
norcocaethylene have been characterized in the present study for the first time, and they are compared with those against
cocaine. According to the obtained kinetic data, wild-type human BChE showed a similar catalytic efficiency against
norcocaethylene (kcat = 9.5 min-1, KM = 11.7 μM, and kcat/KM = 8.12 × 105 M-1 min-1) to that against (-)-
cocaine (kcat = 4.1 min-1, KM = 4.5 μM, and kcat/KM = 9.1 × 105 M-1 min-1). E14-3 and E12-7 showed an improved catalytic activity against
norcocaethylene compared to wild-type BChE. E12-7 showed a 39-fold improved catalytic efficiency against
norcocaethylene (kcat = 210 min-1, KM = 6.6 μM, and kcat/KM = 3.18 × 107 M-1 min-1). It has been demonstrated that E12-7 as an exogenous
enzyme can efficiently metabolize
norcocaethylene in rats.