Jiyun Zhu, Alexandria M. Kemp, Bala C. Chenna, Vivek Kumar, Andrew Rademacher, Sangho Yun, Arthur Laganowsky, Thomas D. Meek
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引用次数: 0
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(kcat/Ka) vs pH for the substrate (Abz)SAVLQ*SGFRK(Dnp)-NH2 and pKi vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of pK2 = 8.2 ± 0.4 and pK1 = 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 pK2 = 7.8 ± 0.1 and pK1 = 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 pK2 = 8.0 and pK1 = 6.5. The steady-state sKIEs were D2O(kcat/Ka) = 0.56 ± 0.05 and D2Okcat = 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.
期刊介绍：
ACS Catalysis is an esteemed journal that publishes original research in the fields of heterogeneous catalysis, molecular catalysis, and biocatalysis. It offers broad coverage across diverse areas such as life sciences, organometallics and synthesis, photochemistry and electrochemistry, drug discovery and synthesis, materials science, environmental protection, polymer discovery and synthesis, and energy and fuels.
The scope of the journal is to showcase innovative work in various aspects of catalysis. This includes new reactions and novel synthetic approaches utilizing known catalysts, the discovery or modification of new catalysts, elucidation of catalytic mechanisms through cutting-edge investigations, practical enhancements of existing processes, as well as conceptual advances in the field. Contributions to ACS Catalysis can encompass both experimental and theoretical research focused on catalytic molecules, macromolecules, and materials that exhibit catalytic turnover.
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