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 changes in substrate KM, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them.
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All raw data have been deposited in the Dryad Database: https://datadryad.org/stash/share/ZBLh5JFV-KjDQjCmqiX1qSFQbWJ3CXsd9Eu-l8rpxkM. Source data are provided with this paper. All reagents are available upon request from the authors.
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We thank S. Banani and A. Rice for constructs, and all members of the Rosen Laboratory, past and present, for helpful advice and discussions. Research was supported by the Howard Hughes Medical Institute, a Paul G. Allen Frontiers Group Distinguished Investigator Award and a grant from the Welch Foundation (no. I-1544, to M.K.R.).
M.K.R. is a founder of Faze Medicines.
Peer review information Nature Chemical Biology thanks Tanja Mittag and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Sensitivity of enhanced condensate activity to KM, substrate concentration, and partition coefficient.
a, Modeled ratio of total reaction rate in a phase separated solution, with and without recruitment of enzyme and substrate to the scaffold (TotalS and TotalUS, respectively, as a function of substrate concentration (plotted as [S]/KM,US) and partition coefficient (PC). Modeled for KM,US = 150 and KM,S = 50, as measured for FRB-polySH33 + polyPRM5, with identical PC values for enzyme and substrate. Modeling assumes simple, hyperbolic Michaelis-Menten kinetics (see Methods). Color scale is a relative representation of the z-axis values and goes from low (blue) to high (red). Inset is a plot of TotalS:TotalUS rate as a function of substrate concentration at a fixed partition coefficient of 50. b, Modeled ratio of TotalS to TotalUS as a function of PC and the change in KM upon recruitment of enzyme and substrate to the scaffold, KM,US/KM,S. Total substrate concentration, [S]T, set to 0.1 * KM,US. c, Same as (b), except [S]T set to 10 * KM,US.
Extended Data Fig. 2 Total scaffold rate can be less than total unscaffolded activity in certain regimes if enzyme partitioning is much less than substrate partitioning.
a-c, Modeled ratio of total reaction rate in a phase separated solution, with and without recruitment of enzyme and substrate to the scaffold as a function of substrate concentration and substrate partition coefficient (PCS). Both reactions have KM = 150. Enzyme partitioning (PCE) is 1 (a), 10 (b), and 100 (c); enzyme concentration, [E] = 0.1[S]. Model based on 0.01 droplet volume fraction.
Extended Data Fig. 3 Droplet rate increases rapidly relative to bulk as a function of partition coefficient.
a, Modeled ratio of droplet and bulk reaction rates as a function of substrate concentration and partition coefficient (PC). Both reactions are scaffolded and have KM = 50. Enzyme partitioning is identical to substrate partitioning, and [E] = 0.1[S]. b, Modeled fractional activity contributed by the droplet phase as a function of substrate concentration and partition coefficient (PC). Conditions same as in (a), with a 0.01 droplet volume fraction.
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Peeples, W., Rosen, M.K. Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. Nat Chem Biol 17, 693–702 (2021). https://doi.org/10.1038/s41589-021-00801-x
Nature Chemical Biology (2021)