2025
Biomacromolecules

Substrate Exclusion Greenlights Physical Autocatalysis of Enzyme Activity in Membraneless Proto-Organelles

Authors:

Tasdiq Ahmed; Adya Verma; Shuichi Takayama

Keywords:

Substrate Exclusion; Membraneless Proto-Organelles; phase separation; condensate formation; enzyme activity

Abstract:

Cells modulate phase separation to control condensate formation, yet how such organelles affect enzyme activity is poorly understood. This paper describes how substrate-excluding, membraneless proto-organelles can increase enzyme mobility while also controlling the extent of reaction acceleration. The model system─an equimolar polyelectrolyte-nucleotide coacervate─allows for probing compositional influence on the activity of the biopolymer-processing enzyme dextranase. Increasing the phase-forming constituent concentrations is sufficient to sustain fast intradroplet dextranase mobility and enhance hydrolysis, even when placed in highly viscous, concentrated regimes of the organelle-excluded substrate dextran. This catalytic uptick is mediated largely by organelle material properties, eschewing the need for effective substrate or enzyme concentration enrichment. Physical analysis reveals that dextranase dimensions fall within the organelle mesh size range and that constituent concentrations induce changes in droplet viscosity. Enzyme diffusion measurements within polymer solutions that mimic the intracellular/prebiotic landscape underscore the catalytic, and potentially evolutionary, advantage of compartments, enabling faster enzyme mobility.