Abstract
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.
Highlights
The amidase signature (AS)1 family was originally identified by primary structure analysis, which revealed a highly conserved serine- and glycine-rich sequence present in several amidases of bacterial and fungal origin [1, 2]
11 amino acids in Fatty acid amide hydrolase (FAAH) were selected for mutagenesis based on their potential ability to participate in acid/base chemistry and/or hydrogen bonding
Several reports have described the mutagenesis of a select number of conserved AS residues in either FAAH [13, 14, 36, 37] or the Rhodococcal J1 amidase [27], these efforts have produced contradictory proposals regarding the nature of the core catalytic components of the AS family
Summary
Generation of FAAH Mutants—FAAH mutants were constructed in the prokaryotic expression vector pTrcHis A (Invitrogen) using the Quickchange procedure (Stratagene) [13]. The cells were sonicated using a 50-watt tip sonicator and centrifuged in a table top ultracentrifuge at 100,000 ϫ g for 1 h in a TLA-100 rotor (Beckman Instruments) The pellet from this spin was resuspended by sonication in 200 l of buffer 2 (20 mM Hepes, pH 7.8, 150 mM NaCl, 10% glycerol, 1% Triton X-100), rocked for 1 h at 4 °C, and centrifuged at 100,000 ϫ g for 1 h. The supernatant from this spin, constituting solubilized FAAH membrane extracts, was collected, and its protein concentration was determined using the Dc protein assay kit (Bio-Rad). The S217A mutant showed no pH or pD dependence and the ratio kH2O/kD2O was pH-independent
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