The role of liquid-liquid phase separation in regulating enzyme activity

Abstract

Liquid-liquid phase separation (LLPS) is now recognized as a common mechanism underlying regulation of enzyme activity in cells. Insights from studies in cells are complemented by in vitro studies aimed at developing a better understanding of mechanisms underlying such control. These mechanisms are often based on the influence of LLPS on the physicochemical properties of the enzyme's environment. Biochemical mechanisms underlying such regulation include the potential for concentrating reactants together, tuning reaction rates, and controlling competing metabolic pathways. LLPS is thus a powerful tool with extensive utilities at the cell's disposal, e.g. for consolidating cell survival under stress or rerouting metabolic pathways in response to the energy state of the cell. Here, we examin the evidence for how LLPS affects enzyme catalysis and begin to understand emerging concepts and expand our understanding of enzyme catalysis in living cells.

Keywords: Biomolecular condensate; Catalysis; Crowding; Membraneless organelles; Metabolism; Stress response.

Figure 1:. Cells use LLPS to control enzyme activity.

A. LLPS can increase the local concentration of reactants, thus increasing rates of product formation. B. LLPS of enzymes within one particular pathway (pathway A) can lead to increased production of a reagent used in other pathways (pathways B and C). The localized production of the reagent results in localization of these enzymes around the condensate (or even their partitioning into the condensate) [35]. C. Selective co-compartmentalization of certain enzymes in a forked metabolic pathway can divert metabolites toward one pathway. Switching between alternate compartmentalization states may be regulated by post-translational modification of phase-separating proteins, e.g. as a result of the energy state of the cell. D. RNA-binding proteins can phase separate with mRNA and control its translation and degradation, locally and globally.

Figure 2:. Biophysical and biochemical effects of phase separation can function to regulate enzyme activity.

A. LLPS results in a unique local environment that differs from the surrounding dilute phase. The solvent properties of condensates can resemble organic solvents, allowing tuning of reaction conditions by LLPS. The resulting increase in viscosity has been shown to reduce enzyme activity due to reduced mobility of reagents. This implies a balance between contrasting effects to realize optimal reaction conditions within condensates [2,22,75,76]. B. The high cooperativity of LLPS means that enzymatic reactions can be switched on or off. By balancing the concentration of enzyme at the threshold of phase separation (likely through translational regulation), activity can be rapidly switched on by a small increase in local concentration, and off again by a subsequent small decrease depending on cellular needs [5,14,50,61]. C. Crowding within a condensate can result in a variety of effects on kinetic activity. Interactions between components in a condensate can result in a reduced rate of product turnover by freezing enzyme dynamics critical to turnover. Non-interacting components can reduce the available space through their excluded volume, concentrating enzymes together with their substrates, resulting in increased turnover. Finally, components which form weak, nonspecific interactions with the substrate and reduce accessibility to the enzyme active site can result in an apparent reduction in enzyme-substrate affinity [74].