Investigating the mechanisms of enzyme diffusion using mass photometry

Abstract

ABSTRACTThe existence of the phenomenon of enhanced enzyme diffusion (EED) has been a topic of debate in recent literature. The majority of experiments confirming the existence of this phenomenon have been conducted using fluorescence correlation spectroscopy (FCS), but an artefact has been found in some of these experiments. There are various proposed mechanisms to explain the origin of EED, such as conformational changes and oligomeric enzyme dissociation. In our study we use mass photometry (MP), a label-free single-molecule interferometric light scattering technique, to investigate the dependence of the oligomeric states of several enzymes on the presence or absence of substrate. The enzymes of interest in this study are catalase, aldolase, alkaline phosphatase and an alcohol oxidase (VAO) the first three of which were previously studied in the context of enhanced enzyme diffusion. We compared the ratios of oligomeric states in the presence and absence of substrate as well as different substrate and inhibitor concentrations. Catalase and aldolase were found to dissociate into smaller oligomers with substrate, while for alkaline phosphatase and VAO, different behaviors were observed. Intriguingly, the changes in oligomeric states in aldolase and catalase are independent of catalysis. Thus, we have identified a possible mechanism which explains the previously observed enhanced diffusion of enzymes by oligomer dissociation through ligand binding.