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

Autor: Omeir RL; Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York, 11794, USA., Arreaza G, Deutsch DG
Jazyk: angličtina
Zdroj: Biochemical and biophysical research communications [Biochem Biophys Res Commun] 1999 Oct 22; Vol. 264 (2), pp. 316-20.
DOI: 10.1006/bbrc.1999.1524
Abstrakt: 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.
(Copyright 1999 Academic Press.)
Databáze: MEDLINE
Externí odkaz: