Abstract
Secreted phospholipases A2 (sPLA2) are water-soluble lipolytic enzymes that act at the interface of organized lipid substrates, where the catalytic step is coupled to various interfacial phenomena as enzyme penetration, solubilisation of reaction products, lateral packing and loss of mechanical stability of organized assemblies of phospholipid molecule, among others. Using the monomolecular film technique, we compared the interfacial properties of crab digestive sPLA(2) (CDPL) with those of the porcine pancreatic one (PPPL). A kinetic study on the surface pressure dependency of the two sPLA(2) was performed using monomolecular films of three different substrates: di C-12-PC (1.2-dilauroyl-sn-glycerol-3-phosphocholine); di C-12-PG (1.2-dilauroyl-sn-glycerol-3-phosphoglycerol) and di C-12-PE (1.2-dilauroyl-sn-glycerol-3-phosphoethanolamine). The use of a substrate in monolayer state, during the catalytic reactions, allows us to monitor the effect of several physicochemical parameters by altering the "quality of interface". The effect of temperature on the hydrolysis rate of these substrates was also checked. Our results show that activities of both phospholipases were affected by the variation of the subphase temperature. CDPL was irreversibly inactivated by p-bromo-phenacyl bromide, the specific inhibitor of sPLA2. The hyperbolic catalytic behaviour observed was coherent with hopping mode of action, one of the two characteristic mechanisms of interfacial catalysis of sPLA2. (C) 2019 Elsevier B.V. All rights reserved.