Enzymes and Liquid-Liquid Phase Separation: A New Era for the Regulation of Enzymatic Activity

Abstract

Liquid-liquid phase separation (LLPS) is recognized as a mechanism for regulation of enzymatic activity. Biochemical mechanisms include concentrating reactants to enhance reaction rates or sequester enzymes and reactants from each other to reduce the reaction rate. On the other hand, LLPS might also regulate the diffusion of small molecules or important parameters for enzymatic activity (such as modulators, macromolecular crowding and changing the media physicochemical features) increasing or decreasing the reaction rate of the enzymes. Furthermore, the co-compartmentalization of specific enzymes can favour or speed up specific metabolic fluxes. Here, we discuss how LLPS contributed to generate a new era for enzyme regulation and the new possible subtle regulation mechanisms still unexplored. enzyme / liquid-liquid phase separation / enzymatic activity / enzyme regulation / macromolecular crowding / cell metabolism 1. Enzymes and liquid-liquid phase separation Enzymes are catalysts able to substantially increase the chemical reactions rate allowing ultimately to sustain life. Enzymes are also characterised by specificity for substrate/s and have an important role in regulating the metabolism of the cells. However, the dynamic motion of the enzymes' during catalysis is one of the most intriguing and still not completely understood events, along with other aspects of protein chemistry 1)-3). The cell is an organised and crowded environment, in which thousands of processes are active at the same time. Cellular organelles spatially organise the metabolism and the biological matter. These compartments can be physically separated by a membrane boundary from the surrounding cellular environment. Also, these organelles can be impermeable to most biological molecules and examples are given by Golgi apparatus, mitochondria, peroxisomes, and endoplasmic retic-ulum 4),5). However, the presence of micro-scale membrane-less liquid-like compartments have been recently reported in the cytoplasm, nucleus and along the membranes 6)-8). Evidence indicates that Liquid-Liquid Phase Separation (LLPS) is important in many biological processes, as the well-documented stress response (with the formation of stress granules), but also for transcription, signalling and metabolism 9). Further evidence associates aberrant forms of LLPS with several human diseases 10). These discoveries describe the LLPS as a new fundamental physicochemical mechanism for organising the biochemistry of the cells. LLPS allows the formation of at least two different phases, one dense phase formed by concentrated biomolecules that usually interact by multivalent interactions (proteins and/or nucleic acids) and the surrounding dilute phase, depleted of biomolecules 11). The process is dynamic and reversible. To drive the formation of a phase-separated solution, the biomolecules must have specific features and reach a threshold concentration of participating biomolecules 12). Recently, it has been widely shown that LLPS plays important role in the regulation of enzymatic activity, providing spatiotemporal control over enzyme function. However, the physicochemical mechanisms that regulate the enzymatic activity and the possible structural changes at molecular level are largely unknown so far. May enzymes be structurally stabi-lised upon formation of LLPS? Or can it be only the more active conformation that is stabilised? In this Review, we will discuss the mechanisms of regulation of enzymatic activity in the membrane-less compartments present in literature and the other possible mechanisms still unexplored. 2. Mechanisms for regulation of the enzymatic activity mediated by LLPS A direct way for the cell to achieve control over space of 生物物理 63（1） ，12-15（2023）