2014 Sep 10. [Epub ahead of print]
Anandamide Hydrolysis in FAAH Reveals a Dual Strategy for Efficient Enzyme-assisted Amide Bond Cleavage via Nitrogen Inversion.
Abstract
Anandamide is one of the main neurotransmitters involved in the endocannabinoid-mediated control of pain. Its biological functions are regulated by the fatty acid amide hydrolase (FAAH) that cleaves anandamide to generate arachidonic acid via a specific mechanism of hydrolytic degradation. Herein, we combined classical molecular dynamics (MD) and quantum mechanical/molecular mechanics (QM/MM) simulations to unravel the whole catalytic cycle of FAAH in complex with its main substrate anandamide. Experimental results suggested a two-step catalytic mechanism, characterized by (1) acyl-enzyme formation after hydrolysis of the substrate amide bond and (2) deacylation reaction with restoration of the catalytic machinery. While microsecond MD simulations of FAAH in a realistic membrane/water environment provided a solid model for the reactant state of the enzymatic complex (Palermo et al. JCTC, 2013), QM/MM simulations depict now a highly concerted mechanism for both the acylation and deacylation chemical steps. We also clarify the unbinding from the catalytic site of the substrate leaving group during enzyme acylation, an essential physical step connecting the acylation and deacylation reactions. We found that a crucial event for anandamide hydrolysis is the inversion of the reactive nitrogen of the scissile amide bond, which occurs during the acylation rate-limiting step. While we quantitatively describe the energetic cost for nitrogen inversion, we show that 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 catalytic site, such as the conserved oxyanion hole and the H-bond acceptor Met191, are key actors in a novel enzymatic strategy that uses the combination of the two canonical “H-bond” and “proton-shuttle” mechanisms for enzyme-assisted amide bond hydrolysis. This new strategy is likely to be of general applicability to other amidases/peptidases that show similar catalytic site architectures, providing crucial insights for de-novo enzyme design or drug discovery efforts.
- PMID:
- 25205244
- [PubMed – as supplied by publisher]
