Slow diffusion limits phosphorylation in a biomolecular condensate
Biomolecular condensates form dynamic compartments that regulate biochemical reactions in cells. Condensates recruit many kinases and regulate their enzymatic activity. Condensates alter the rate of enzymatic reactions through several opposing effects, so it is unclear whether these mostly enhance or retard phosphorylation. Here, we use a synthetic condensate formed by intrinsically disordered proteins to show that slow diffusion in the condensate controls phosphorylation kinetics in the dense phase. We vary the length of substrates by appending phase-separating repeat proteins of different lengths, in order to study how phosphorylation depends on partitioning, diffusion and volume fraction across substrate motifs with different intrinsic kinetics. The condensate environment is generally inhibitory to phosphorylation, although the enzyme remains intact. This inhibition is partially offset by an enhanced reaction rate in the dilute phase, likely due to soluble nanoclusters. Phosphorylation rates are strongly correlated to diffusion coefficients of substrates in the condensate, suggesting mass-transport limitation. Our results suggest that condensates can modify the substrate usage of a kinase via different trade-offs between diffusion and partitioning. We suggest that diffusion limitations are likely a common feature of many macromolecular reactions in condensates, and that high fluidity is crucial for condensates to act as reaction crucibles.