Abstract
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(k (cat)/K (a)) vs pH for the substrate (Abz)SAVLQ*SGFRK(Dnp)-NH(2) and pK (i) vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of pK (2) = 8.2 ± 0.4 and pK (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 pK (2) = 7.8 ± 0.1 and pK (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 pK values for the cysteine and histidine groups of pK (2) = 8.0 and pK (1) = 6.5. The steady-state sKIEs were (D(2)O)(k (cat)/K (a)) = 0.56 ± 0.05 and (D(2)O) k (cat) = 1.0 ± 0.1, and sKIEs indicated that the Cys-S(-)-HisH(+) tautomer was enriched in D(2)O. Presteady-state kinetic analysis of (Abz)SAVLQ*SGFRK(Dnp)-NH(2) exhibited transient lags preceding steady-state rates, which were considerably faster in D(2)O than in H(2)O. The transient rates encompass steps that include substrate binding and acylation, and are faster in D(2)O wherein the more active Cys-S(-)-HisH(+) tautomer predominates. A full catalytic mechanism for 3CL-PR is proposed.