Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme 
responsible for the hydrolysis of a number of neuromodulatory fatty acid amides, 
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 stretch of 
approximately 130 amino acids termed the "amidase signature sequence." Recently, 
site-directed mutagenesis studies of FAAH have targeted a limited number of 
conserved residues in the amidase signature sequence of the enzyme, identifying 
Ser-241 as the catalytic nucleophile and Lys-142 as an acid/base catalyst. The 
roles of several other conserved residues with potentially important and/or 
overlapping catalytic functions have not yet been examined. In this study, we 
have mutated all potentially catalytic residues in FAAH that are conserved among 
members of the amidase signature family, and have assessed their individual 
roles in catalysis through chemical labeling and kinetic methods. Several of 
these residues appear to serve primarily structural roles, as their mutation 
produced FAAH variants with considerable catalytic activity but reduced 
expression in prokaryotic and/or eukaryotic systems. In contrast, five 
mutations, K142A, S217A, S218A, S241A, and R243A, decreased the amidase activity 
of FAAH greater than 100-fold without 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 particular interest, one mutant, R243A, displayed 
uncompromised esterase activity but severely reduced amidase activity, 
indicating that the amidase and esterase efficiencies of FAAH can be 
functionally uncoupled. Collectively, these studies provide evidence that 
amidase signature enzymes represent a large class of serine-lysine catalytic 
dyad hydrolases whose evolutionary distribution rivals that of the catalytic 
triad superfamily.