RT Journal Article SR Electronic T1 Phase Separation Can Increase Enzyme Activity by Concentration and Molecular Organization JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.09.15.299115 DO 10.1101/2020.09.15.299115 A1 William Peeples A1 Michael K. Rosen YR 2020 UL http://biorxiv.org/content/early/2020/09/17/2020.09.15.299115.abstract AB 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.Competing Interest StatementM.K.R. is a founder of Faze Medicines