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, includin g anandamide, 2-arachidonyl glycerol, and oleamide, This activity is inhibi ted by serine and cysteine reactive agents, suggesting that the active site contains a serine or cysteine residue. Therefore serine and cysteine resid ues were mutated to alanine and the effects on activity were determined. Mu tants were prepared using site-directed mutagenesis methods and expressed i n COS-7 cells. Serine mutations S217A and S241A completely abolished enzyma tic activity. Mutants S152A and C249A had no effect on activity, while S218 A showed a slight decrease in activity. To confirm these results biochemica lly, the mutant enzymes were reacted with the irreversible inhibitor [C-14] -diisopropyl fluorophosphate. All of the mutants except S217A and S241A wer e 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 enzy me activity. (C) 1999 Academic Press.