Kinetic characterization of cholinesterases and a therapeutically valuable cocaine hydrolase for their catalytic activities against heroin and its metabolite 6-monoacetylmorphine.

As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases, including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental conditions. It has been demonstrated that AChE and BChE have similar k values (2100 and 1840 min, respectively) against heroin, but with a large difference in K (2170 and 120 μM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (k = 2150 min and K = 245 μM) and 6-MAM (k = 0.223 min and K = 292 μM), with relatively larger K values and lower catalytic efficiency compared to BChE. Notably, the K values of CocH1 against both heroin and 6-MAM are all much larger than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.