Interfacial mechanism of phospholipase A2: pH-dependent inhibition and Me-beta-cyclodextrin activation.
The pH-dependent activity of phospholipase A(2) (PLA(2)) from Naja mossambica mossambica venom and the membrane-water partitioning of the lipid hydrolysis products were investigated in solid-supported palmitoyl-oleyl-phosphatidylcholine-d(31) (POPC-d(31)) membranes using neutron reflection. At pH 5, PLA(2) interacts only weakly with the substrate membrane and leads to no observable membrane breakdown, which is consistent with protonation of the catalytic histidine (His48, pK(a) approximately 6.2). The rate of the lyso-lipid partitioning into the solution phase is the same at pH 9 as at pH 7.4, and the relative membrane-water partitioning of the products is essentially the same; that is, the fatty acid accumulates in the membrane, and only the lyso-lipid is solubilized. However, Me-beta-cyclodextrin (Me-beta-CD) activates PLA(2) irrespective of pH by facilitating the solubilization of the lyso-lipid product, but not the fatty acid, of which only 22% is encapsulated at pH 9. Since no product solubilization is observed at pH 5 in the absence of Me-beta-CD, this suggests that the hydrolytic mechanism of PLA(2) is not fully disabled at pH 5 but is inhibited by a mechanism, which is counteracted by Me-beta-CD-mediated release of the lyso-lipid. Me-beta-CD does not interact with the substrate membrane, which indicates that at low pH the product extraction occurs directly from the enzyme active site outside the immediate membrane-water interface, whereas at pH 7-9, direct solubilization of the lyso-lipid from the membrane can also contribute to activation of PLA(2).
Wacklin
Hanna P
HP
Australian Nuclear Science and Technology Organisation, PMB1, Menai, NSW 2234, Australia. wacklin@ill.eu
D000818:Animals; D002384:Catalysis; D004546:Elapid Venoms / Q000201:enzymology*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D033363:Neutron Diffraction / Q000379:methods; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2 / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000378:metabolism*
D004546:Elapid Venoms; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D064801:Phospholipase A2 Inhibitors; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D054467:Phospholipases A2; C028694:1-palmitoyl-2-oleoylphosphatidylcholine