Identification of two serine residues involved in catalysis by fatty acid amide hydrolase.

Fatty acid amide hydrolase is an integral membrane protein that hydrolyzes a novel and growing class of neuromodulatory fatty acid molecules, including anandamide, 2-arachidonyl glycerol, and oleamide. This activity is inhibited by serine and cysteine reactive agents, suggesting that the active site contains a serine or cysteine residue. Therefore serine and cysteine residues were mutated to alanine and the effects on activity were determined. Mutants were prepared using site-directed mutagenesis methods and expressed in COS-7 cells. Serine mutations S217A and S241A completely abolished enzymatic activity. Mutants S152A and C249A had no effect on activity, while S218A showed a slight decrease in activity. To confirm these results biochemically, the mutant enzymes were reacted with the irreversible inhibitor (14)C]-diisopropyl fluorophosphate. All of the mutants except S217A and S241A were labeled. We therefore confirm that fatty acid amide hydrolase is a serine hydrolase and propose that both Ser-217 and Ser-241 are essential for enzyme activity.