title: Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α.
abstract: Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.
authors.affiliation: Department of Mechanistic Biochemistry, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Pedicord
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Flynn
authors.forename: Caroline
authors.initials: C
authors.lastname: Fanslau
authors.forename: Maricar
authors.initials: M
authors.lastname: Miranda
authors.forename: Lisa
authors.initials: L
authors.lastname: Hunihan
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Robertson
authors.forename: Bradley C
authors.initials: BC
authors.lastname: Pearce
authors.forename: Xuan-Chuan
authors.initials: XC
authors.lastname: Yu
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Westphal
authors.forename: Yuval
authors.initials: Y
authors.lastname: Blat
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002863:Chromogenic Compounds / Q000737:chemistry; D003512:Cyclohexanones / Q000737:chemistry; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D007783:Lactones / Q000737:chemistry; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D000077403:Orlistat; D013379:Substrate Specificity
chemical_list: D002863:Chromogenic Compounds; D003512:Cyclohexanones; D007783:Lactones; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; D000077403:Orlistat; C480280:DAGLA protein, human; D008071:Lipoprotein Lipase