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Updated: Jun 4, 2025

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity
Published on: August 19, 2013
Catalytic Mechanism of SARS-CoV-2 3-Chymotrypsin-Like Protease as Determined by Steady-State and Pre-Steady-State
Jiyun Zhu1, Alexandria M Kemp1, Bala C Chenna1
1Departments of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States.
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)-NH2 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 D2O(k cat/K a) = 0.56 ± 0.05 and D2O k 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.
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