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( / ) vs pH for the substrate (Abz)SAVLQSGFRK(Dnp)-NH and p vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of p = 8.2 ± 0.4 and p = 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 = 7.8 ± 0.1 and p = 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 values for the cysteine and histidine groups of p = 8.0 and p = 6.5. The steady-state sKIEs were ( / ) = 0.56 ± 0.05 and = 1.0 ± 0.1, and sKIEs indicated that the Cys-S-HisH tautomer was enriched in DO. Presteady-state kinetic analysis of (Abz)SAVLQSGFRK(Dnp)-NH exhibited transient lags preceding steady-state rates, which were considerably faster in DO than in HO. The transient rates encompass steps that include substrate binding and acylation, and are faster in DO wherein the more active Cys-S-HisH tautomer predominates. A full catalytic mechanism for 3CL-PR is proposed.