Abstract
Carboxylesterase-1 (CE-1) is a crucial enzyme responsible for metabolism/activation/inactivation of xenobiotics (therapeutic agents, prodrugs, abused drugs, and organophosphorus nerve agents etc.) and also involved in many other biological processes. In this study, we performed extensive computational modeling and simulations to understand the fundamental reaction mechanism of cocaine hydrolysis catalyzed by CE-1, revealing that CE-1-catalyzed cocaine hydrolysis follows a novel reaction pathway with only two reaction steps: a single-step acylation process and a single-step deacylation process. In the transition states of both single-step processes, the cocaine NH group joins the oxyanion hole to form an additional hydrogen bond with the negatively charged carbonyl oxygen atom of the cocaine. Thus, the transition states are stabilized by both intermolecular and intramolecular hydrogen bonds with the methyl ester of cocaine, specifically the carbonyl oxygen atom. The rate-limiting transition state is associated with the acylation process, and the activation free energy barrier was predicted to be 20.1 kcal/mol. Further, in vitro experimental kinetic analysis was performed for human CE-1-catalyzed cocaine hydrolysis. For CE-1-catalyzed cocaine hydrolysis, the computationally predicted free energy barrier (20.1 kcal/mol) is reasonably close to the experimentally derived turnover number ( k(cat) = 0.058 min(-1)), indicating the reasonability of the computational results. The obtained novel mechanistic insights are expected to benefit not only CE-1 related rational drug discovery but also future research on the catalytic mechanism of other esterases.