Catalytic Mechanism of SARS-CoV-2 3-Chymotrypsin-Like Protease as Determined by Steady-State and Pre-Steady-State Kinetics

The 3-chymotrypsin-like protease (3CL-PR; also known as Main protease) of SARS-CoV-2 is a cysteine protease that is the target of the COVID-19 drug, Paxlovid. Here, we report for 3CL-PR, the pH-rate profiles of a substrate, an inhibitor, affinity agents, and solvent kinetic isotope effects (sKIEs) obtained under both steady-state and pre-steady-state conditions. "Bell-shaped" plots of log(<i>k</i> _cat/<i>K</i> _a) vs pH for the substrate (Abz)SAVLQ*SGFRK(Dnp)-NH₂ and p<i>K</i> _i vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of p<i>K</i> ₂ = 8.2 ± 0.4 and p<i>K</i> ₁ = 6.2 ± 0.3, respectively, are required for catalysis, and the pH-dependence of inactivation of 3CL-PR by iodoacetamide and diethylpyrocarbonate identified enzymatic groups of p<i>K</i> ₂ = 7.8 ± 0.1 and p<i>K</i> ₁ = 6.05 ± 0.07, which must be unprotonated for maximal inactivation. These data are most consistent with the presence of a neutral catalytic dyad (Cys-SH-His) in the 3CL-PR free enzyme, with respective p<i>K</i> values for the cysteine and histidine groups of p<i>K</i> ₂ = 8.0 and p<i>K</i> ₁ = 6.5. The steady-state sKIEs were ^D₂O(<i>k</i> _cat/<i>K</i> _a) = 0.56 ± 0.05 and ^D₂O <i>k</i> _cat = 1.0 ± 0.1, and sKIEs indicated that the Cys-S^--HisH⁺ tautomer was enriched in D₂O. Presteady-state kinetic analysis of (Abz)SAVLQ*SGFRK(Dnp)-NH₂ exhibited transient lags preceding steady-state rates, which were considerably faster in D₂O than in H₂O. The transient rates encompass steps that include substrate binding and acylation, and are faster in D₂O wherein the more active Cys-S^--HisH⁺ tautomer predominates. A full catalytic mechanism for 3CL-PR is proposed.