Clarifying the catalytic roles of conserved residues in the amidase signature family

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme r esponsible for the hydrolysis of a number of neuromodulatory fatty acid ami des, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide, FAAH belongs to a large class of hydrolytic enzymes termed the "amidase signature family," whose members are defined by a conserved s tretch of approximately 130 amino acids termed the "amidase signature seque nce." Recently, site-directed mutagenesis studies of FAAH have targeted a l imited number of conserved residues in the amidase signature sequence of th e enzyme, identifying Ser-241 as the catalytic nucleophile and Lys-142 as a n acid/base catalyst. The roles of several other conserved residues with po tentially important and/or overlapping catalytic functions have not yet bee n examined. In this study, we have mutated all potentially catalytic residu es in FAAH that are conserved among members of the amidase signature family , and have assessed their individual roles in catalysis through chemical la beling and kinetic methods. Several of these residues appear to serve prima rily structural roles, as their mutation produced FAAH variants with consid erable catalytic activity but reduced expression in prokaryotic and/or euka ryotic systems. In contrast, five mutations, K142A, S217A, S218A, S241A, an d R243A, decreased the amidase activity of FAAH greater than 100-fold witho ut detectably impacting the structural integrity of the enzyme, The pH rate profiles, amide/ester selectivities, and fluorophosphonate reactivities of these mutants revealed distinct catalytic roles for each residue, Of parti cular interest, one mutant, R243A, displayed uncompromised esterase activit y but severely reduced amidase activity, indicating that the amidase and es terase efficiencies of FAAH can be functionally uncoupled. Collectively, th ese studies provide evidence that amidase signature enzymes represent a lar ge class of serine-lysine catalytic dyed hydrolases whose evolutionary dist ribution rivals that of the catalytic triad superfamily.