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Article Dans Une Revue Biochimie Année : 2014

Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid–water interface

Résumé

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.
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hal-03272282 , version 1 (06-12-2022)

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Paternité - Pas d'utilisation commerciale - Pas de modification

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Anaïs Bénarouche, Vanessa Point, Frédéric Carrière, Jean-François Cavalier. Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid–water interface. Biochimie, 2014, 101, pp.221-231. ⟨10.1016/j.biochi.2014.01.019⟩. ⟨hal-03272282⟩

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