Publications

• Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.

  Cravatt B.F., Giang D.K., Mayfield S.P., Boger D.L., Lerner R.A., Gilula N.B.

  Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the s ... >> More

  Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates. << Less

  Nature 384:83-87(1996) [PubMed] [EuropePMC]

• Molecular characterization of human and mouse fatty acid amide hydrolases.

  Giang D.K., Cravatt B.F.

  Recently, we reported the isolation, cloning, and expression of a rat enzyme, fatty acid amide hydrolase (FAAH), that degrades bioactive fatty acid amides like oleamide and anandamide to their corresponding acids, thereby serving to terminate the signaling functions of these molecules. Here, we re ... >> More

  Recently, we reported the isolation, cloning, and expression of a rat enzyme, fatty acid amide hydrolase (FAAH), that degrades bioactive fatty acid amides like oleamide and anandamide to their corresponding acids, thereby serving to terminate the signaling functions of these molecules. Here, we report the molecular characterization of both a mouse and a human FAAH and compare these enzymes to the rat FAAH. The enzymes are well conserved in primary structure, with the mouse and rat FAAHs sharing 91% amino acid identity and the human FAAH sharing 82% and 84% identity with the rat FAAH and mouse FAAH, respectively. In addition, the expressed human and rat FAAHs behave biochemically as membrane proteins of comparable molecular size and show similar, but distinguishable, enzymological properties. The identification of highly homologous FAAH proteins in rat, mouse, and human supports a general role for the fatty acid amides in mammalian biology. << Less

  Proc. Natl. Acad. Sci. U.S.A. 94:2238-2242(1997) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• A second fatty acid amide hydrolase with variable distribution among placental mammals.

  Wei B.Q., Mikkelsen T.S., McKinney M.K., Lander E.S., Cravatt B.F.

  Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydr ... >> More

  Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species. << Less

  J. Biol. Chem. 281:36569-36578(2006) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Structural adaptations in a membrane enzyme that terminates endocannabinoid signaling.

  Bracey M.H., Hanson M.A., Masuda K.R., Stevens R.C., Cravatt B.F.

  Cellular communication in the nervous system is mediated by chemical messengers that include amino acids, monoamines, peptide hormones, and lipids. An interesting question is how neurons regulate signals that are transmitted by membrane-embedded lipids. Here, we report the 2.8 angstrom crystal str ... >> More

  Cellular communication in the nervous system is mediated by chemical messengers that include amino acids, monoamines, peptide hormones, and lipids. An interesting question is how neurons regulate signals that are transmitted by membrane-embedded lipids. Here, we report the 2.8 angstrom crystal structure of the integral membrane protein fatty acid amide hydrolase (FAAH), an enzyme that degrades members of the endocannabinoid class of signaling lipids and terminates their activity. The structure of FAAH complexed with an arachidonyl inhibitor reveals how a set of discrete structural alterations allows this enzyme, in contrast to soluble hydrolases of the same family, to integrate into cell membranes and establish direct access to the bilayer from its active site. << Less

  Science 298:1793-1796(2002) [PubMed] [EuropePMC]

• Reversible hydrolysis and synthesis of anandamide demonstrated by recombinant rat fatty-acid amide hydrolase.

  Kurahashi Y., Ueda N., Suzuki H., Suzuki M., Yamamoto S.

  Previously we suggested that one porcine brain enzyme (anandamide amidohydrolase) catalyzed both the hydrolysis of anandamide and its synthesis from arachidonic acid and ethanolamine (Ueda et al., J. Biol. Chem. 270, 23823-23827, 1995). In the present study we investigated the reversibility of the ... >> More

  Previously we suggested that one porcine brain enzyme (anandamide amidohydrolase) catalyzed both the hydrolysis of anandamide and its synthesis from arachidonic acid and ethanolamine (Ueda et al., J. Biol. Chem. 270, 23823-23827, 1995). In the present study we investigated the reversibility of the enzyme reactions by the use of recombinant fatty-acid amide hydrolase of rat liver, which appears to be catalytically identical to porcine anandamide amidohydrolase. The particulate fraction of the COS-7 cells, in which the rat enzyme was overexpressed, hydrolyzed anandamide with a specific activity of 132 nmol/min/mg protein at 37 degrees C, and the Km value for anandamide was 18 microM. The enzyme also synthesized anandamide at a rate of 177 nmol/min/mg protein, and the Km values for arachidonic acid and ethanolamine as substrates were as high as 190 microM and 36 mM, respectively. The control cells transfected with the insert-free vector showed neither the hydrolase activity nor the synthase activity. Thus, the hydrolase and synthase are attributed to the same enzyme protein coded by one gene. However, the enzyme may act as a hydrolase rather than a synthase under physiological conditions judging from its high Km values for substrates in the synthase reactions. In addition, primary amides of fatty acids such as arachidonamide and oleamide and fatty acid ester like methyl arachidonate were hydrolyzed at considerable rates, and their reverse reactions occurred even if at lower rates. << Less

  Biochem. Biophys. Res. Commun. 237:512-515(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase.

  Vila A., Rosengarth A., Piomelli D., Cravatt B., Marnett L.J.

  Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variet ... >> More

  Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH. << Less

  Biochemistry 46:9578-9585(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Evidence for distinct roles in catalysis for residues of the serine-serine-lysine catalytic triad of fatty acid amide hydrolase.

  McKinney M.K., Cravatt B.F.

  Fatty acid amide hydrolase (FAAH) is a mammalian amidase signature enzyme that inactivates neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The recent determination of the three-dimensional structures of FAAH and two dist ... >> More

  Fatty acid amide hydrolase (FAAH) is a mammalian amidase signature enzyme that inactivates neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The recent determination of the three-dimensional structures of FAAH and two distantly related bacterial amidase signature enzymes indicates that these enzymes employ an unusual serine-serine-lysine triad for catalysis (Ser-241/Ser-217/Lys-142 in FAAH). Mutagenesis of each of the triad residues in FAAH has been shown to severely reduce amidase activity; however, how these residues contribute, both individually and in cooperation, to catalysis remains unclear. Here, through a combination of site-directed mutagenesis, enzyme kinetics, and chemical labeling experiments, we provide evidence that each FAAH triad residue plays a distinct role in catalysis. In particular, the mutation of Lys-142 to alanine indicates that this residue functions as both a base involved in the activation of the Ser-241 nucleophile and an acid that participates in the protonation of the substrate leaving group. This latter property appears to support the unusual ability of FAAH to hydrolyze amides and esters at equivalent rates. Interestingly, although structural evidence indicates that the impact of Lys-142 on catalysis probably occurs through the bridging Ser-217, the mutation of this latter residue to alanine impaired catalytic activity but left the amide/ester hydrolysis ratios of FAAH intact. Collectively, these findings suggest that FAAH possesses a specialized active site structure dedicated to a mechanism for competitive amide and ester hydrolysis where nucleophile attack and leaving group protonation occur in a coordinated manner dependent on Lys-142. << Less

  J. Biol. Chem. 278:37393-37399(2003) [PubMed] [EuropePMC]