Mechanistic dissection of increased enzymatic rate in a phase-separated compartment

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 K-M. 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 changes in substrate K-M, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them.

Automated summary

Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane, leading to accelerated reaction rates through increased concentrations and physical organization of molecules. The SUMOylation enzyme cascade can be recruited into droplets generated by liquid-liquid phase separation, significantly increasing reaction rates with substantial specificity among different substrates. This modulation of reaction rates in condensates is achieved through mass action and changes in substrate K-M, likely due to scaffold-induced molecular organization.