Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 May;14(5):2349-2357.
doi: 10.1016/j.apsb.2024.03.001. Epub 2024 Mar 6.

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

Rabea Voget  1 , Julian Breidenbach  1 , Tobias Claff  1 , Alexandra Hingst  1 , Katharina Sylvester  1 , Christian Steinebach  1 , Lan Phuong Vu  1 , Renato H Weiße  2 , Ulrike Bartz  3 , Norbert Sträter  2 , Christa E Müller  1 , Michael Gütschow  1
Affiliations

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

Rabea Voget et al. Acta Pharm Sin B. 2024 May.

Abstract

A titrant for the SARS-CoV-2 main protease (Mpro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active Mpro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of Mpro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the Mpro-catalyzed cleavage of these substrates were determined.

Keywords: Active-site titration; COVID-19; Fluorogenic substrates; Inner filter effect; Main protease; Peptide nitriles; SARS-CoV-2; X-ray crystallography.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Image 1
Graphical abstract
Scheme 1
Scheme 1
Synthesis of the active-site titrant 1.
Figure 1
Figure 1
Co-crystal structure of 1 with SARS-CoV-2 Mpro (PDB ID: 8QDC, chain A). (A) Covalent binding of 1 to the Mpro active site and accessible subsites. The (2Fo‒Fc)-type electron density of the ligand and Cys145 is shown as yellow mesh (contoured at 1 σ). (B) 2D interaction diagram of 1 with Mpro.
Figure 2
Figure 2
Active-site titration of His-tagged SARS-CoV-2 Mpro (2.0 ng/μL) with 1 under use of substrate 2 (50 μmol/L). The data are means from five independent experiments performed with aliquots from the same enzyme preparation. The equivalent point, obtained by linear regression as the x-axis intercept, corresponded to an active enzyme concentration of 32.6 ± 3.8 nmol/L and a proportion of 56% active enzyme. The standard deviations refer to the linear regression.
Figure 3
Figure 3
Determination of kinetic parameters for conversion of substrates 2–5 by His-tagged SARS-CoV-2 Mpro. Data are means of triplicate measurements. (A, D) IFE-corrected fluorescence was translated into product concentration. (B, C) IFE correction was not implemented. (A) Determination of kcat, kcat/Km, and Km values of substrate 2. (B) Determination of the kcatapp value of substrate 3. (C) Determination of the kcatapp value of substrate 4. (D) Determination of kcat, kcat/Km, and Km values of substrate 5.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Lu R., Zhao X., Li J., Niu P., Yang B., Wu H., et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395:565–574. - PMC - PubMed
    1. Hoffmann M., Kleine-Weber H., Schroeder S., Krüger N., Herrler T., Erichsen S., et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181:271–280. - PMC - PubMed
    1. V’kovski P., Kratzel A., Steiner S., Stalder H., Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol. 2021;19:155–170. - PMC - PubMed
    1. Zhang L., Lin D., Sun X., Curth U., Drosten C., Sauerhering L., et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020;368:409–412. - PMC - PubMed
    1. Hu Q., Xiong Y., Zhu G.H., Zhang Y.N., Zhang Y.W., Huang P., et al. The SARS-CoV-2 main protease (Mpro): structure, function, and emerging therapies for COVID-19. MedComm. 2022;3:e151. - PMC - PubMed

LinkOut - more resources

-