Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling

Area of Science:

• Biochemistry
• Origin of Life Studies
• Materials Science

Background:

• Membraneless organelles are crucial for cellular function and early life evolution.
• Understanding enzyme interactions within coacervates is key to deciphering substrate dynamics.
• Synthetic coacervates offer a model system to investigate enzyme-substrate relationships.

Purpose of the Study:

• To investigate how the molecular crowding within coacervates influences enzyme-substrate interactions.
• To quantify the kinetics of a protease acting on peptide substrates within a coacervate system.
• To develop enzyme-responsive coacervates for studying biological processes.

Main Methods:

• Oligopeptide-based coacervates (anionic Asp-peptide and cationic Arg-peptide) with a cleavage site were synthesized.
• Enzyme kinetics were modeled using Cy5.5-labeled peptides and a self-quenching mechanism.
• The specificity constant (kcat/KM) was determined for the protease (Mpro) within the coacervate.

Main Results:

• Coacervates dissolved in the presence of the main protease (Mpro).
• The specificity constant (kcat/KM) was determined to be 5817 M-1 s-1, comparable to free substrates.
• Enzyme kinetics were found to be dependent on the type and quantity of incorporated dye.

Conclusions:

• A simple design for enzyme-responsive coacervates was developed.
• Insights into enzyme-coacervate interactions and substrate dynamics were provided.
• This work contributes to understanding the role of compartmentalization in early biological systems.