Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

Liquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using L-lactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electrostatic interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold de-crease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increas-ing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the drop-lets provides essential information about enzyme function in living cells in addition to biotechnology applications.