Thermodynamic principles of enzymatic regulation in biomolecular condensates from reaction-coupled molecular modeling

Biomolecular condensates are dynamic cellular assemblies stabilized by weak intermolecular interactions. Cells regulate condensate formation, composition, and function through energy-consuming processes such as post-translational modifications (PTMs), which modify the physicochemical properties of condensate components, thereby dynamically reshaping these interactions. Here, we investigate how enzymatic reactions regulate phase-separated systems using a thermodynamically consistent particle-based model, which allows sampling of out-of-equilibrium steady states. We find that reaction kinetics are intrinsically coupled to the local molecular environment, leading to the formulation of two general principles. First, reactions that weaken favorable interactions are thermodynamically suppressed within condensates. As a consequence, regulation of condensate solubility is most efficient when PTMs tune interactions to values close to the solubility threshold. In contrast, reaction rates are generally enhanced at condensate interfaces, where the thermodynamic inhibition is relieved, but reactant availability remains high. Higher-resolution simulations of FUS and DDX4 proteins indicate that this interfacial contribution remains substantial even for micron-sized condensates, positioning interfaces as key determinants of biochemical activity in phase-separated systems alongside the properties of the condensate bulk. Together, these findings identify general, thermodynamic principles that govern the regulation of biomolecular condensates and link enzymatic activity with phase separation. Moreover, they provide a thermodynamically consistent molecular framework that can be applied to a broad range of regulatory processes in active phase-separated systems.