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

Citation
Rl. Omeir et al., Identification of two serine residues involved in catalysis by fatty acid amide hydrolase, BIOC BIOP R, 264(2), 1999, pp. 316-320
Citations number
52
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISSN journal
0006291X → ACNP
Volume
264
Issue
2
Year of publication
1999
Pages
316 - 320
Database
ISI
SICI code
0006-291X(19991022)264:2<316:IOTSRI>2.0.ZU;2-W
Abstract
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.