Modulation of phospholipase A2: identification of an inactive membrane-bound state

Phospholipase A2-catalyzed hydrolysis of vesicular phospholipid has been used to model the modulation of an enzyme's function by membrane properties. Phospholipase A2's (PLA2) kinetics toward large unilamellar vesicles (LUV) composed of dipalmitoylphosphatidylcholine (DPPC) are anomalous; these is a slow initial phase of catalysis (a lag) which ends abruptly with a sudden increase in the catalytic rate (a burst). The sudden increase in activity due to the accumulation of a critical mole fraction of reaction products and substrate undergoes compositional phase separation. In this work, we address the molecular details of the coupling between compositional phase separation and activation of PLA2. A prominent model for this coupling is that compositional phase separation leads to a surface for which PLA2 has increased affinity, resulting in the recruitment of PLA2 from solution to the surface. Here, we show that the bulk of PLA2 is associated with the membrane at a time well before the abrupt increase in catalytic rate. This finding indicates that there must be a relatively inactive, membrane-bound state. Furthermore, PLA2's kinetics are anomalous even when the substrate comprises a surface to which PLA2 is bound throughout the time course. With DPPC LUV as the substrate, detailed time courses show that the description of the time course as a lag and a burst is inadequate. Instead, the time course consists of multiple phases of acceleration and deceleration. The data presented here suggest that all these various changes in catalytic rate may be due to product-induced changes in membrane properties. In particular, we suggest that nonequilibrium, microheterogeneities of lipid composition may underlie these very complicated kinetics.