Interfacial catalysis by human 85 kDa cytosolic phospholipase A2 on anionic vesicles in the scooting mode.
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.
Bayburt
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Department of Chemistry, University of Washington, Seattle 98195-1700, USA.
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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
D008051:Lipid Bilayers; D010743:Phospholipids; D011994:Recombinant Proteins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2