Charge-Mediated Interactions Affect Enzymatic Reactions in Peptide Condensates

Biomolecular condensates, formed through liquid-liquid phase separation (LLPS), serve as enzymatic reaction centers in cells by increasing local concentrations of enzymes and substrates, thereby facilitating reaction kinetics and regulatory mechanisms. Inspired by these natural systems, synthetic condensates are being developed for diverse applications, including payload delivery, sensing, and as microreactors where enzymatic reaction kinetics can be modulated by factors like pH, viscosity, and enzyme-substrate co-localization. Here, we investigate how the physicochemical properties of enzymes and substrates influence condensate formation and function as microreactors. Focusing on cellulase and alkaline phosphatase, which differ in molecular weight and isoelectric point, we employed a minimalistic complex coacervation system of oppositely charged LLPS-promoting peptides. Our findings show how electrostatic forces within condensates influence their role as microreactors. Specifically, the ability of condensates to encapsulate or exclude phosphatase, cellulase, and their substrates, which is pivotal for the regulation of reaction kinetics, is determined by the enzyme surface charge, substrate charge, and condensate charge stoichiometry. These results highlight the potential of utilizing electrostatic forces within condensates to modulate enzymatic reactions, providing critical insights for developing synthetic condensates as microreactors in biotechnology and materials science.