Anandamide is one of the main neurotransmitters involved in the endocannabinoid-mediated control of pain. It was the first endogenous cannabinoid to be discovered. Its biological functions are primarily regulated by the fatty acid amide hydrolase (FAAH) enzyme, which cleaves anandamide with a specific mechanism of hydrolytic degradation. The inhibition of FAAH, therefore, increases the level of endogenous anandamide, which is a promising strategy to treat pain and several inflammatory-related pathologies. Herein, we combined classical molecular dynamics (MD) and mixed quantum mechanical/molecular mechanics (QM/MM) simulations to unravel the whole catalytic cycle of FAAH in complex with its main substrate anandamide. On the basis of experimental results, a two-step mechanism was proposed for anandamide hydrolysis, which is characterized by (1) acyl-enzyme formation after hydrolysis of the substrate amide bond and (2) deacylation reaction with restoration of the catalytic machinery. Our findings depict a highly concerted mechanism for both the acylation and deacylation chemical steps. We show that simultaneous proton transfers, needed to facilitate nucleophilic attack and product formation, characterize each step of this complex multi-event reaction. Importantly, we have also quantitatively described the inversion of the reactive nitrogen of the scissile amide bond in anandamide, which is a crucial event during the acylation rate-limiting step. FAAH uses an exquisite catalytic strategy to induce amide bond distortion, reactive nitrogen inversion, and amide bond hydrolysis, promoting catalysis to completion. Structural features of the FAAH catalytic site, such as the oxyanion hole, are key to efficient nitrogen inversion and amide bond hydrolysis during the acylation reaction. This enzymatic strategy is likely to be of general applicability to other amidases/peptidases that show similar catalytic site architectures. Moreover, the detailed knowledge and dynamic description of this critical event for efficient enzyme-assisted amide bond hydrolysis might help in de novo enzyme design or drug discovery efforts.

Hydrolysis of anandamide in FAAH shows that nitrogen inversion is key to efficient enzyme-assisted amide bond cleavage

248th National Meeting (2014)

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