The N-terminal cysteine hydrolase N-acylethanolamine acid amidase (NAAA) catalyzes the hydrolytic deactivation of the lipid messenger palmitoylethanolamide (PEA), with optimal activity at acidic pH. Using the crystal structure of human NAAA as a starting point, we investigated the catalytic mechanism of PEA hydrolysis with a multiscale approach based on classic molecular dynamics (MD) and quantum mechanical/molecular mechanics (QM/MM) simulations coupled with enhanced sampling and path-collective variables (PCVs). The proton configuration of the catalytic nucleophile, Cys126, and of the surrounding carboxylates was critical to preserve the active site architecture. A stable Michaelis complex was then used to reconstruct the free-energy surfaces of NAAA acylation and deacylation during PEA hydrolysis. Acylation emerged as the critical step, with Cys126 acting both as an acid, to protonate the ethanolamine leaving group, and as a nucleophile, to attack the PEA carbonyl carbon. The ethanol fragment of PEA did not appear to play an indispensable role in acylation, a result further supported by kinetic experiments showing that NAAA hydrolyzes palmitoyl methyl amide (PMA) with high catalytic efficiency. Our multiscale approach identified a distinctive protonation state and catalytic mechanism for NAAA which accounts for pH-dependent activity, mutagenesis data, and mechanism of covalent inhibitors.
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