Linking Phospholipase Mobility to Activity by Single‐Molecule Wide‐Field Microscopy

Many of the biological processes taking place in cells are mediated by enzymatic reactions occurring in the cell membrane. Understanding interfacial enzymatic catalysis is therefore crucial to the understanding of cellular function. Unfortunately, a full picture of the overall mechanism of interfacial enzymatic catalysis, and particularly the important diffusion processes therein, remains unresolved. Herein we demonstrate that single‐molecule wide‐field fluorescence microscopy can yield important new information on these processes. We image phospholipase enzymes acting upon bilayers of their natural phospholipid substrate, tracking the diffusion of thousands of individual enzymes while simultaneously visualising local structural changes to the substrate layer. We study several enzyme types with different affinities and catalytic activities towards the substrate. Analysis of the trajectories of each enzyme type allows us successfully to correlate the mobility of phospholipase with its catalytic activity at the substrate. The methods introduced herein represent a promising new approach to the study of interfacial/heterogeneous catalysis systems.