N-Acylethanolamine Acid Amidase (NAAA): Mechanism of Palmitoylethanolamide Hydrolysis Revealed by Mechanistic Simulations

Abstract

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.

UPLC-MS/MS Method for Analysis of Endocannabinoid and Related Lipid Metabolism in Mouse Mucosal Tissue

“…Moreover, macrophages express the FAEmetabolizing enzyme, NAAA, which in addition to FAAH, may contribute to FAE hydrolysis in lung (Tsuboi et al, 2007). Nonetheless, we report a near full inhibition of mouse lung FAE hydrolysis with URB597; however, our methods and conditions described above for assaying activity of FAAH differed from those reported for assaying activity of NAAA and may contribute to differential effects for inhibitors under assay conditions that favor FAAH over NAAA activity (e.g., differing centrifugation speeds) (Solorzano et al, 2009;Scalvini et al, 2020). It is also notable that the UPLC/MS/MS assays we describe here utilize tissue homogenates that, in contrast to assays using purified enzymes, contain a variety of enzymes in addition to DGL, MGL, and AHD6 that may contribute to hydrolysis of corresponding substrates.…”

Different roles for the acyl chain and the amine leaving group in the substrate selectivity of N-Acylethanolamine acid amidase

“…We prepared and tested FAE species with acyl chains C15:1D10 (7), C16:1D9 (8), C17:1D10 (9), C18:1D9 (OEA, 10), C19:1D10 (11) and C20:1D9 (12) (Figure 2(D,E)). FAE C17:1D10 was the best NAAA substrate in this series (Figure 2(D,E)) and displayed a catalytic efficiency that was superior to both shorter-chain (7,8) and longer-chain compounds (10)(11)(12) including OEA (Figure 2(F)). Of note, all monounsaturated FAEs exhibited significantly lower V max / K m ratios compared to PEA (Table 1).…”

“…We modelled the catalytic cysteine in its neutral amine-anionic thiolate form and the adjacent aspartic acid (Asp150) with its carboxylic group in neutral state and engaged in an H-bond with the neutral form of the enzyme’s N -terminal amine. The assignment of the protonation state of ionisable residues was driven by the results of our previous work showing that a supervised proton distribution is fundamental for the dynamic stability of hNAAA 10 . The catalytic site and its surroundings are highly conserved in human, rabbit and rat NAAA, and the protein stability likely relies on a proton distribution within the catalytic residue and the acid residues in proximity of the active site that is similar across different species.…”

“…Consistently with the maximum of activity of NAAA observed at pH 4.5, all the histidine and basic residues were modelled in their protonated form. The protonation state for Cys131 and its surroundings was assigned as described in a recent study for our group, where a specific protonation pattern was found to provide the greatest stability during molecular dynamics simulations of human NAAA 10 . Cys131 was modelled in its neutral amine-anionic thiolate form, while the protonation state of acidic residues within 10 Å of the catalytic cysteine was assigned as follows: Asp150, Asp199, Glu200 and Asp304, conserved in both hNAAA and ocNAAA, were modelled in their neutral form.…”

“…In this structure, the polar head of PEA accommodates within a solvent exposed cleft comprising the catalytic residue, with the amide group making polar interactions with the oxyanion hole residues (Asn292 and Glu195 in hNAAA) and with the backbone of Asp145. Following this hypothesis, the mechanism of PEA hydrolysis catalysed by NAAA has been recently investigated through QM/MM simulations 10 . According to the proposed mechanism, during the acylation the catalytic cysteine, stabilised by Asp145, acts both as an acid and as a nucleophile, by protonating the amide nitrogen of PEA and favouring the expulsion of the ethanolamine fragment as leaving group upon the formation of a thioester bond.…”