An enzyme caught in action: Direct imaging of hydrolytic function and domain formation of phospholipase A 2 in phosphatidylcholine monolayers

Abstract

Phospholipase A 2, a ubiquitous lipolytic enzyme that actively catalyses hydrolysis of phospholipids, has been studied as a model for enzyme-substrate reactions, as a membrane structural probe, and as a model for lipid-protein interactions. Its mechanism of action remains largely controversial. We report here for the first time direct microscopic observation of the lipolytic action of fluorescently marked phospholipase A 2 ( Naja naja naja) against phosphatidylcholine monolayers in the lipid phase transition region. Under these conditions, phospholipase A 2 is shown to target and hydrolyse solid-phase lipid domains of L-α-dipalmitoylphosphatidylcholine. In addition, after a critical extent of monolayer hydrolysis, the enzyme itself aggregates into regular, visible proteinaceous domains within the lipid monolayer. Solid-phase lipid hydrolysis indicates a preferential hydrolytic environment for phospholipase A 2 while enzyme domain formation points to a possible allosteric inhibition mechanism by hydrolysis products.