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The present work describes the mechanism of FAAH inhibition by BIA 10\u20102474 as a target\u2010specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme\u2018s active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on\u2010site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme\u2018s catalytic nucleophile to form a covalent enzyme\u2010inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10\u20102474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre\u2010orienting and stabilizing the inhibitor in a near\u2010reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.<\/jats:p>","DOI":"10.1002\/cbic.202200166","type":"journal-article","created":{"date-parts":[[2022,7,17]],"date-time":"2022-07-17T22:19:29Z","timestamp":1658096369000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Inactivation Mechanism of the Fatty Acid Amide Hydrolase Inhibitor BIA 10\u20102474"],"prefix":"10.1002","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0342-7424","authenticated-orcid":false,"given":"Nuno M. F. S. A.","family":"Cerqueira","sequence":"first","affiliation":[{"name":"Department of Research &amp; Development BIAL, Portela &amp; C\u00aa. S.A.  S. Mamede do Coronado Portugal"}]},{"given":"Marco","family":"Neves","sequence":"additional","affiliation":[{"name":"Department of Research &amp; Development BIAL, Portela &amp; C\u00aa. S.A.  S. Mamede do Coronado Portugal"}]},{"given":"Juliana","family":"Rocha","sequence":"additional","affiliation":[{"name":"BioSIM Department of Biomedicine Faculty of Medicine University of Porto  Porto Portugal"}]},{"given":"Patr\u00edcio","family":"Soares\u2010da Silva","sequence":"additional","affiliation":[{"name":"Department of Research &amp; Development BIAL, Portela &amp; C\u00aa. S.A.  S. Mamede do Coronado Portugal"},{"name":"Department of Biomedicine Unit of Pharmacology &amp; Therapeutics Faculty of Medicine University of Porto  Porto Portugal"},{"name":"MedInUP \u2013 Center for Drug Discovery and Innovative Medicines University of Porto  Porto Portugal"}]},{"given":"P. Nuno","family":"Palma","sequence":"additional","affiliation":[{"name":"Department of Research &amp; Development BIAL, Portela &amp; C\u00aa. S.A.  S. 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