Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A2 hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA2 shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA2, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d31-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA2 molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA2.