Dynamic Interchange Between Two Protonation States is Characteristic of Active Sites of Cholinesterases

Cholinesterases are well-known and widely studied enzymes crucial to human health and involved in neurology, Alzheimer, and lipid metabolism. The protonation pattern of cholinesterases’ active sites influences all the chemical processes within, including reaction, covalent inhibition by nerve agents, and reactivation. Our understanding of it is, however, limited. In this study, we used enhanced-sampling quantum-mechanical/molecular-mechanical calculations to show that cholinesterases mostly function as dynamical mixtures of two protonation states. Proton transfer between two non-catalytic glutamate residues occurs by Grotthuss mechanism via a mediator water molecule. We show that this uncovered complexity of active sites presents a challenge for classical molecular dynamics simulations that calls for special treatment. The proton transfer barrier of 1.65 kcal/mol opens the discussion on potential existence of two conjugated low barrier hydrogen bonds in the inhibited form of butyrylcholinesterase. These findings expand our understanding of structural features expressed by highly evolved enzymes and guide future advances in cholinesterase-related protein- and drug design studies.