PHOSPHOLIPID ACTIVATION OF COBRA VENOM PHOSPHOLIPASE-A2 .2. CHARACTERIZATION OF THE PHOSPHOLIPID-ENZYME INTERACTION

Activation of cobra venom phospholipase A2 toward phosphatidylethanolamine by phosphatidylcholine in mixed micelles has been suggested to be caused by a direct phosphatidylcholine-enzyme interaction. Comparable activation of phospholipase A2 toward phosphatidylethanolamine also occurs with nonsubstrates sphingomyelin and lysophosphatidylcholine. Phospholipids with anionic head groups and water-soluble phosphorylcholine or glycerol phosphorylcholine alone are nonactivators. Hydrolysis of phosphatidylethanolamine is 0.06 that of phosphatidylcholine, and the rate increases abruptly (0.38) when two methyl groups are present on the nitrogen. The hydrolysis of all phospholipids examined is enhanced dramatically in the presence of activator lipid. The activation profile for phosphatidylethanolamine hydrolysis in the presence of sphingomyelin parallels cosubstrate phosphatidylcholine activation. Phospholipid binding studies reveal a similar apparent KD (1 mM) for all phospholipids examined. Enzyme inactivation by p-bromophenacyl bromide, a reagent which inactivates the enzyme through modification of a histidine in the active site, is significantly protected by lipid substrates and fatty acids, while nonsubstrate activator lipids do not protect. The pKa controlling phosphatidylethanolamine hydrolysis was found to shift from 6.5 to 5.8 in response to phosphatidylcholine activation of the enzyme, suggesting that the activator lipid causes a conformational change in the enzyme. These results can be explained by the existence of two binding sites: (i) an activator site which requires a lipid molecule containing the phosphorylcholine moiety and a fatty acyl chain and (ii) a head-group nonspecific catalytic site. © 1979, American Chemical Society. All rights reserved.