Phase Separation Can Increase Enzyme Activity by Concentration and Molecular Organization

Abstract

Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid-liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and by changes in substrate KM, likely due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and by physically organizing them.

A framework for understanding the functions of biomolecular condensates across scales

“…2a). It has recently been reported that in a biochemical reconstitution of the SUMOylation cascade in engineered condensates, when two substrates were present, only the one recruited into the condensates was SUMOylated efficiently 50 . However, in this system, rate enhancement and specificity were constrained by the properties of enzymes and substrates, in manners that should be general.…”

“…For example, the rel ative change in reaction rate due to association with a condensate depended on the K M value for each individual substrate: substrates present at concentrations well above their K M value exhibited little change in the reaction rate upon condensate formation because the enzyme was already saturated even in the initial homogeneous solution; by contrast, when substrates were present at concentrations well below their K M value, formation of condensates produced strong reaction rate enhance ment. Thus, reaction rates for specific substrates can be enhanced upon condensate formation, whereas for others these rates will change only marginally, dictated by the substrate concentrations, K M values and degree of enrichment in the dense phase 50 . These considera tions may be important in understanding specificity of pathways such as RNAi, where thousands of different substrates can compete for the same enzyme.…”

“…In addition to the dwell time, the molecular organ ization imparted by condensate scaffolds can further enhance activity 50 . Specifically, scaffolds can tether the substrate and enzyme in close proximity, and in con sequence decrease the K M value of the reaction.…”

Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation

“…Organizing cellular processes into biomolecular condensates is a mechanism to regulate biochemistry in distinct ways: altering molecular conformation and organization to promote specificity, increasing local concentration to accelerate enzymatic activity, and coupling interactions to enforce reaction directionality 1 – 10 . The emergent properties afforded by formation of extensive interaction networks in condensates can also enhance enzymatic activity beyond local concentration effects 11 , 12 .…”

Controlling biomolecular condensates via chemical reactions

“…Biomolecular condensates are small droplets that structure the cell interior of eukaryotes [1,2] and prokaryotes [3][4][5]. They form by phase separation and participate in a wide range of cellular functions [6]: since they are chemically distinct from their surroundings, they can act as reaction centres [7,8], like the nucleolus inside the nucleus [9]. In particular, locally elevated concentrations can induce polymerization, as in microtubule branching [10] or in centrosomes [11,12], which additionally control the subcellular organization.…”