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

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• 作者：Anaï ; s Bé ; narouche¹ ; Vanessa Point¹ ; Fré ; dé ; ric Carriè ; re ; ^{carriere@imm.cnrs.fr" class="auth_mail} ; Jean-Franç ; ois Cavalier ; ^{jfcavalier@imm.cnrs.fr" class="auth_mail}
• 关键词：Enzyme kinetics ; Interfacial enzymology ; Lipase ; Lipolysis ; Monomolecular film ; Lipase inhibition
• 刊名：Biochimie
• 出版年：June, 2014
• 年：2014
• 卷：101
• 期：Complete
• 页码：221-231
• 全文大小：1888 K

文摘

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 k_cat/K_M*聽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.