Influence of product phase separation on phospholipase A(2) hydrolysis of supported phospholipid bilayers studied by force microscopy

In situ atomic force microscopy studies reveal a marked influence of the initial presence of hydrolysis products on the hydrolysis of supported phospholipid bilayers by phospholipase, A(2). By analysis of the nano-scale topography of a number of supported bilayers, with different initial product concentrations, made by Langmuir-Blodgett deposition, we show that small depressions enriched in products are efficiently promoting enzyme degradation of the bilayer. These small depressions, which are indicative of phase separation, are initially present in samples with 75% products. The kinetics of phospholipase A(2) exhibit under certain conditions an initial phase of slow hydrolysis, termed the latency phase, followed by a marked increase in the hydrolysis rate. The appearance of the phase-separated bilayer is strikingly similar to that of bilayers; at the end of the latency phase. By analysis of individual nano-scale defects we illustrate a quantitative difference in the growth rates of defects caused by product aggregation and other structural defects. This difference shows for the first time how the enzyme prefers one type of defect to another.