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

Abstract

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 Llactate 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 electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing 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 droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

Protein conformation and biomolecular condensates

“…Selectivity is based in the porous nature and excluded volume effects of the condensates, which act like dynamic noncovalently crosslinked meshes of homo and heterotypic interactions. Reactivity is mainly based in the law of mass action, although molecular conformation, scaffold-induced molecular organizations, and changes in diffusion rates also modify reactivity ( André and Spruijt, 2020 ; Ura et al., 2020 ; Peeples and Rosen, 2021 ; Zhang et al., 2021 ).…”

Condensate Formation by Metabolic Enzymes in Saccharomyces cerevisiae

“…For protein condensates using liquid-liquid phase separation, Peeple and Rosen suggested in a preprint that condensate formation increases the cascade reaction activities by SUMOylation enzymes on high K M substrates but not on low K M substrates, with identical k cat [43]. Ura and colleagues reported in a preprint that in vitro condensate formation of lactate oxidase drastically increased k cat and reduced K M [45], suggesting that there is something more occurring other than the effect on substrate channeling by enzyme condensates. This suggests that the enzyme concentration by liquid-liquid phase separation has a somewhat similar effect to the previously known immobilization of the enzyme.…”

“…It is important to elucidate the effect of large-scale enzyme assembly on cell physiology, including metabolism, methods to control the association and dissociation of condensates by metabolic enzymes. Moreover, there is a need to determine the kinetics of each enzyme in the condensate, as enzyme kinetics in condensates can change drastically [45] and can affect parameters such as those used for metabolic flux analysis. These would provide the apparent activity of condensed enzymes at bulk concentrations, thereby estimating the effect on the whole cell.…”