Abstract
The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM 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.
Highlights
The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM)
Sedimentation velocity analytical ultracentrifugation (SV-AUC), which precludes dilution during the experiment, at concentrations ranging from 18–90 μM clearly shows that MProM is mainly monomeric with no detectable dimer form
The results presented above indicate that MProM adopts a tertiary fold like MProWT as shown by its catalytic activity and ability to bind GC373 and form a transition state analog at the active site, and by the CD spectrum
Summary
The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Despite the monomer form adopting a native-like tertiary fold, as shown for various mutations or deletions in the sequence, monomeric variants of MPro are reported to exhibit very low or no catalytic activity[7,17,21,26–33]. This has been attributed to a collapsed active site which impairs the binding of. In the monomeric structure the loop comprising residues S139 to L141 was shown to transform into a 310-helix such that the rearranged N142 interacting with E166 blocks entry to the S1 subsite This is consistent with the observation that mutation E166A impairs substrate binding[17,20]
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