title: 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 gamma-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.
authors.affiliation: Department of Chemistry, University of Washington, Seattle 98195-1700, USA.
authors.forename: T
authors.initials: T
authors.lastname: Bayburt
authors.forename: M H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000138:chemical synthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D008051:Lipid Bilayers; D010743:Phospholipids; D011994:Recombinant Proteins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2