Catalytic Mechanism\nof SARS-CoV‑2 3‑Chymotrypsin-Like\nProtease as Determined by Steady-State and Pre-Steady-State Kinetics

The 3-chymotrypsin-like protease (3CL-PR; also known\nas Main protease)\nof SARS-CoV-2 is a cysteine protease that is the target of the COVID-19\ndrug, Paxlovid. Here, we report for 3CL-PR, the pH-rate profiles of\na substrate, an inhibitor, affinity agents, and solvent kinetic isotope\neffects (sKIEs) obtained under both steady-state and pre-steady-state\nconditions. “Bell-shaped” plots of log(<i>k</i><sub>cat</sub>/<i>K</i><sub>a</sub>) vs pH for the substrate\n(Abz)SAVLQ*SGFRK(Dnp)-NH<sub>2</sub> and p<i>K</i><sub>i</sub> vs pH for a peptide aldehyde inhibitor demonstrated that essential\nacidic and basic groups of p<i>K</i><sub>2</sub> = 8.2 ±\n0.4 and p<i>K</i><sub>1</sub> = 6.2 ± 0.3, respectively,\nare required for catalysis, and the pH-dependence of inactivation\nof 3CL-PR by iodoacetamide and diethylpyrocarbonate identified enzymatic\ngroups of p<i>K</i><sub>2</sub> = 7.8 ± 0.1 and p<i>K</i><sub>1</sub> = 6.05 ± 0.07, which must be unprotonated\nfor maximal inactivation. These data are most consistent with the\npresence of a neutral catalytic dyad (Cys-SH-His) in the 3CL-PR free\nenzyme, with respective p<i>K</i> values for the cysteine\nand histidine groups of p<i>K</i><sub>2</sub> = 8.0 and\np<i>K</i><sub>1</sub> = 6.5. The steady-state sKIEs were <sup>D<sub>2</sub>O</sup>(<i>k</i><sub>cat</sub>/<i>K</i><sub>a</sub>) = 0.56 ± 0.05 and <sup>D<sub>2</sub>O</sup><i>k</i><sub>cat</sub> = 1.0 ± 0.1, and sKIEs indicated that\nthe Cys-S<sup>–</sup>-HisH<sup>+</sup> tautomer was enriched\nin D<sub>2</sub>O. Presteady-state kinetic analysis of (Abz)SAVLQ*SGFRK(Dnp)-NH<sub>2</sub> exhibited transient lags preceding steady-state rates, which\nwere considerably faster in D<sub>2</sub>O than in H<sub>2</sub>O.\nThe transient rates encompass steps that include substrate binding\nand acylation, and are faster in D<sub>2</sub>O wherein the more active\nCys-S<sup>–</sup>-HisH<sup>+</sup> tautomer predominates. A\nfull catalytic mechanism for 3CL-PR is proposed.