Characterization and manipulation of the acyl chain selectivity of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme t hat catabolizes several neuromodulatory fatty acid amides, including the en dogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large group of hydrolytic enzymes termed the amidase signatur e (AS) family that is defined by a conserved, linear AS sequence of approxi mately 130 amino acids. Members of the AS family display strikingly differe nt substrate selectivities, yet the primary structural regions responsible for defining substrate recognition in these enzymes remain unknown. In this study, a series of unbranched p-nitroanilide (pNA) substrates ranging from 6 to 20 carbons in length was used to probe the acyl chain binding specifi city of FAAH, revealing that this enzyme exhibits a strong preference for a cyl chains 9 carbons in length or longer. A fluorophosphonate inhibitor of FAAH containing a photoactivatable benzophenone group was synthesized and u sed to locate a region of the enzyme implicated in substrate binding. Prote ase digestion and mass spectrometry analysis of FAAH-inhibitor conjugates i dentified the major site of cross-linking as residues 487-493. Site-directe d mutagenesis revealed that a single residue in this region, 1491, strongly influenced substrate specificity of FAAH. For example, an I491A mutant dis played a greatly reduced binding affinity for medium-chain pNA substrates ( 7-12 carbons) but maintained nearly wild-type binding and catalytic constan ts for longer chain substrates (14-20 carbons). Mutation of 1491 to aromati c or more polar residues generated enzymes with relative hydrolytic efficie ncies for medium versus long-chain pNAs that varied up to 90-fold. Collecti vely, these studies indicate that 1491 participates in hydrophobic binding interactions with medium-chain FAAH substrates. Additionally, the significa nt changes in substrate selectivity achieved by single amino acid changes s uggest that FAAH possesses a rather malleable substrate binding domain and may serve, along with other AS enzymes, as a template for the engineering o f amidases with novel and/or tailored specificities.