Metal-dependent and metal-free mechanisms of peptide condensate catalysts

Abstract Condensates formed via liquid-liquid phase separation (LLPS) provide a chemically versatile environment for catalysis through dynamic molecular interactions. We present designed biomolecular condensates, formed by LLPS of minimalistic histidine-containing peptides, catalyzing ester hydrolysis with two distinct mechanisms. Zn 2+ -dependent condensates activate a coordinating water molecule at the active site, formed by Zn 2+ -histidine coordination, enabling nucleophilic attack. We show that dense-phase basicity, internal mobility, and Zn 2+ accumulation within the condensates collectively govern their catalytic activity. In the absence of Zn 2+ , catalysis is driven by intermolecular low-barrier hydrogen bonds between histidine residues, facilitating nucleophile formation. Combined computational and experimental evidence reveals the molecular basis of these catalytic pathways, demonstrating the functionality of biomolecular condensates in catalysis and nanotechnology. These findings establish a foundation for exploring mechanisms of metal-free emergent catalysis within complex liquid assemblies, expanding the potential of LLPS-based systems in green chemistry and advanced materials.