title: 2,3-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol is a substrate for human glucocerebrosidase.
abstract: Glucocerebrosidase, the lysosomal enzyme that is deficient in patients with Gaucher's disease, hydrolyses non-physiological aryl beta-D-glucosides and glucocerebroside, its substrate in vivo. We document that 2,3,-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol (2,3,-di-14:0-beta-Glc-DAG) inhibits human placental glucocerebrosidase activity in vitro (Ki 0.18 mM), and the nature of inhibition is typical of a mixed-type pattern. Furthermore, 2,3-di-14:0-beta-Glc-DAG was shown to be an excellent substrate for the lysosomal beta-glucosidase (Km 0.15 mM; Vmax. 19.8 units/mg) when compared with the natural substrate glucocerebroside (Km 0.080 mM; Vmax. 10.4 units/mg). The observations that (i) glucocerebrosidase-catalysed hydrolysis of 2,3-di-14:0-beta-Glc-DAG is inhibited by conduritol B epoxide and glucosylsphingosine, and (ii) spleen and brain extracts from patients with Gaucher's disease are unable to hydrolyse 2,3-di-14:O-beta-Glc-DAG demonstrate that the same active site on the enzyme is responsible for catalysing the hydrolysis of 4-methylumbelliferyl beta-D-glucopyranoside, glucocerebroside and 2,3-di-14:O-beta-Glc-DAG. With the aid of computer modelling we have established that the oxygen atoms in 2,3-DAG-Glc at the C-1, C-4*, C-5* (the ring oxygen in glucose) and C-2 positions correspond topologically to the oxygens at C-1, C-4* and C-5* and the nitrogen atom attached to C-2 respectively in glucocerebroside (* signifies a carbon atom in glucose); furthermore, all of the distances with respect to overlap of corresponding heteroatoms range from 0.02 A to 0.77 A (0.002-0.077 nm). A root-mean-square deviation of 0.31 A (0.031 nm) was obtained when the energy-minimized structures of 2,3-di-14:O-beta-Glc-DAG and glucocerebroside were compared using the latter four heteroatom co-ordinates.
authors.affiliation: Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque 87131.
authors.forename: R H
authors.initials: RH
authors.lastname: Glew
authors.forename: V
authors.initials: V
authors.lastname: Gopalan
authors.forename: C A
authors.initials: CA
authors.lastname: Hubbell
authors.forename: R V
authors.initials: RV
authors.lastname: Devraj
authors.forename: R A
authors.initials: RA
authors.lastname: Lawson
authors.forename: W F
authors.initials: WF
authors.lastname: Diven
authors.forename: D A
authors.initials: DA
authors.lastname: Mannock
mesh_terms: D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002151:Calorimetry; D003198:Computer Simulation; D005260:Female; D005776:Gaucher Disease / Q000201:enzymology; D005962:Glucosylceramidase / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism; D006017:Glycolipids / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D012016:Reference Values; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000378:metabolism
chemical_list: D005963:Glucosylceramides; D006017:Glycolipids; C051386:1,2-di-O-tetradecyl-3-O-(glucopyranosyl)glycerol; D001617:beta-Glucosidase; D005962:Glucosylceramidase