Chemical and mutagenic investigations of fatty acid amide hydrolase: Evidence for a family of serine hydrolases with distinct catalytic properties

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible fo r the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase si gnature family". At present, this enzyme family's catalytic mechanism remai ns poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic m ethods. In particular, we focused on the respective roles of three serine r esidues that are conserved in all amidase signature enzymes (S217, S218, an d S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218 A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S 217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), support ing independent catalytic functions for these serine residues. Affinity lab eling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphsnate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K-m implicated a base involve d in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH 's conserved histidines (H184, H358, and H449) generated active enzymes, in dicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mech anism.