Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MPro WT ) was assessed using a predominantly monomeric mutant (MPro M ). Rates of MPro WT and MPro M catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MPro WT , to MPro M leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.