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 p K i vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of p K 2 = 8.2 0.4 and p K 1 = 6.2 0.3, respectively, are required for catalysis, and the pH-dependence of inactivation of 3CL-PR by iodoacetamide and diethylpyroc... More
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 p K i vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of p K 2 = 8.2 0.4 and p K 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 K 2 = 7.8 0.1 and p K 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 K values for the cysteine and histidine groups of p K 2 = 8.0 and p K 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.
