title: Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid-water interface.
abstract: The lipolysis reaction carried out by lipases at the water-lipid interface is a complex process including enzyme conformational changes, adsorption/desorption equilibrium and substrate hydrolysis. Mixed monomolecular films of the lipase inhibitor Orlistat and 1,2-dicaprin were used here to investigate the adsorption of dog gastric lipase (DGL) followed by the hydrolysis of 1,2-dicaprin. The combined study of these two essential catalysis steps was made possible thanks to the highest affinity of DGL for Orlistat than 1,2-dicaprin and the fact that the inhibition of DGL by Orlistat is reversible. Upon DGL binding to mixed 1,2-dicaprin/Orlistat monolayers, an increase in surface pressure reflecting lipase adsorption was first recorded. Limited amounts of Orlistat allowed to maintain DGL inactive on 1,2-dicaprin during a period of time that was sufficient to determine DGL adsorption and desorption rate constants. A decrease in surface pressure reflecting 1,2-dicaprin hydrolysis and product desorption was observed after the slow hydrolysis of the covalent DGL-Orlistat complex was complete. The rate of 1,2-dicaprin hydrolysis was recorded using the surface barostat technique. Based on a kinetic model describing the inhibition by Orlistat and the activity of DGL on a mixed 1,2-dicaprin/Orlistat monolayer spread at the air-water interface combined with surface pressure measurements, it was possible to monitor DGL adsorption at the lipid-water interface and substrate hydrolysis in the course of a single experiment. This allowed to assess the kcat/KM* ratio for DGL acting on 1,2-dicaprin monolayer, after showing that mixed monolayers containing a low fraction of Orlistat were similar to pure 1,2-dicaprin monolayers.
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
authors: Anaïs
authors: A
authors: Bénarouche
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
authors: Vanessa
authors: V
authors: Point
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Electronic address: carriere@imm.cnrs.fr.
authors: Frédéric
authors: F
authors: Carrière
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Electronic address: jfcavalier@imm.cnrs.fr.
authors: Jean-François
authors: JF
authors: Cavalier
mesh_terms: D000327:Adsorption; D000818:Animals; D004075:Diglycerides / Q000737:chemistry; D004285:Dogs; D006868:Hydrolysis; D007700:Kinetics; D007783:Lactones / Q000737:chemistry*; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry*; D008958:Models, Molecular; D000077403:Orlistat; D013270:Stomach / Q000201:enzymology; D013499:Surface Properties; D014867:Water / Q000737:chemistry
chemical_list: D004075:Diglycerides; D007783:Lactones; D008055:Lipids; D014867:Water; C044297:1,2-didecanoylglycerol; D000077403:Orlistat; D008049:Lipase
keywords: Enzyme kinetics; Interfacial enzymology; Lipase; Lipase inhibition; Lipolysis; Monomolecular film