Enzyme kinetics model for the coronavirus main protease including dimerization and ligand binding

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• The coronavirus main protease (MPro) is essential for viral replication and a key target for SARS-CoV-2 antivirals.
• Some MPro enzymatic activities show biphasic concentration response curves (CRCs), with activation at low and inhibition at high ligand concentrations.
• Previous explanations involving ligand-induced dimerization lacked quantitative kinetic modeling.

Purpose of the Study:

• To develop and validate a quantitative kinetic model for MPro enzymatic activity that integrates dimerization and ligand binding.
• To elucidate the molecular mechanisms underlying biphasic CRCs in MPro.
• To characterize the binding and kinetic effects of an inhibitor (GC376) and a substrate on MPro.

Main Methods:

• Development of a novel kinetic model incorporating dimerization and ligand binding.
• Global fitting of the model to diverse biochemical and biophysical data using Bayesian regression.
• Analysis of the effects of the inhibitor GC376 and a fluorescent peptide substrate on MPro activity and dimerization.

Main Results:

• The developed kinetic model successfully explained MPro biphasic CRCs.
• The inhibitor GC376 strongly induced MPro dimerization and bound to the dimer without cooperativity.
• The fluorescent peptide substrate had minimal impact on dimerization but exhibited positive cooperativity upon binding to the dimer.
• Differential effects on dimerization and binding cooperativity explain the observed biphasic behavior.

Conclusions:

• The study provides a quantitative mechanistic explanation for MPro biphasic CRCs, linking it to ligand-specific effects on dimerization and substrate turnover.
• Understanding these kinetics is vital for designing effective SARS-CoV-2 antiviral strategies targeting MPro.
• The model offers a framework for analyzing other enzymes exhibiting complex concentration-dependent behaviors.