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.
Omeir
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RL
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York, 11794, USA.
Arreaza
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Deutsch
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D000409:Alanine / Q000737:chemistry; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D007531:Isoflurophate; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010741:Phospholipases A / Q000378:metabolism; D011480:Protease Inhibitors; D012694:Serine / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D000099252:Fatty Acid Amide Hydrolases
D000409:Alanine; D000581:Amidohydrolases; D003545:Cysteine; D007531:Isoflurophate; D010741:Phospholipases A; D011480:Protease Inhibitors; D012694:Serine; D013438:Sulfhydryl Compounds; D000099252:Fatty Acid Amide Hydrolases