Interfacial Catalysis by Human 85 kDa Cytosolic Phospholipase A2 on Anionic Vesicles in the Scooting Mode

Abstract

Analysis of phospholipases A₂ on model phospholipid bilayers in which enzyme is essentially irreversibly bound at the lipid−water interface, termed “scooting mode”, is a useful tool for studying the kinetic properties of interfacial enzymes. It is shown that human cytosolic 85 kDa phospholipase A₂ (cPLA₂) hydrolyzes sn-2-arachidonyl-containing phospholipids or the γ-linolenoyl ester of 7-hydroxycoumarin (GLU) dispersed in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (L-DOPM) in the scooting mode. Trapping of cPLA₂ on L-DOPM vesicles is rapid and independent of product formation. Slowing of cPLA₂-catalyzed hydrolysis of substrates present in phosphatidylmethanol and phosphatidylcholine vesicles is primarily due to apparent inactivation rather than to substrate depletion. cPLA₂ phosphorylated on serine 505 by mitogen-activated protein kinase displays a 30% increase in the rate of sn-2-arachidonylphosphatidylcholine hydrolysis in the scooting mode compared to that of the nonphosphorylated enzyme. Kinetic parameters of cPLA₂ acting on a variety of different phosphatidylmethanol vesicles were evaluated, and the results are discussed in terms of active site affinities for substrates and of lateral organization of substrates in the bilayer. A key result is that the sigmoidal kinetics reported previously using 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles are most prominent near the phase transition temperature of DMPM. No sigmoidal kinetics was observed using L-DOPM vesicles. The results of kinetic experiments and the behavior of a fluorescent substrate analog are consistent with nonideal mixing of substrate in DMPM vesicles, but not in L-DOPM vesicles, suggesting that apparent saturation and sigmoidal kinetics are more a result of nonideal mixing of substrate in DMPM vesicles than of active site binding of substrate. The fluorescence assay described using L-DOPM/GLU vesicles is useful for evaluating the interfacial behavior of cPLA₂, including its substrate preferences and the effect of active site-directed inhibitors.