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. 2022 Jan 25;12:1299. doi: 10.1038/s41598-022-05424-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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SARS-CoV-2 3CL^pro P108S is declined its enzymatic activity by structural alteration. (a) Recombinant WT or P108S of SARS-CoV2 3CL^pro were analysed with SDS-PAGE visualizing using CBB staining. (b) The enzymatic activities of SARS-CoV2 3CL^pro WT (circle) and P108S (square) were determined using a FRET-based substrate with the cleavage site of SARS CoV-2 3CL^pro (Dabcyl-KTSAVLQ↓SGFRKME-Edans). Error bars show mean ± SD (n = 3). (c) The kinetic parameters of enzyme activity of 3CL^pro WT and P108S were determined using GraphPad Prism 8 software by initial rate measurement of the substrate cleavage. The Kcat/Km value of the P108S mutant enzyme was 42% of that of the WT enzyme, showing 58% reduction. CI indicates 95% confidence interval. (d) Inhibitory activities of SARS-CoV2 3CL^pro WT and P108S by GC376 were analyzed using a FRET-based cleavage assay. The graph shows the relative enzymatic activity. The inhibitory constant (Ki) was calculated using GraphPad Prism 8 software. Error bars show mean ± SD (n = 3). SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; 3CL^pro, 3 chymotrypsin-like protease; WT, Wuhan-strain type; P108S, Pro108Ser-strain type; SDS-PAGE, Sodium dodecyl sulfate–Polyacrylamide gel electrophoresis; CBB, Coomassie Brilliant Blue; FRET, fluorescence resonance energy transfer.