Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics
Rabea Voget, Julian Breidenbach, Tobias Claff, Alexandra Hingst, Katharina Sylvester, Christian Steinebach, Lan Phuong Vu, Renato H.‐J. Weiße, Ulrike Bartz, Norbert Sträter, Christa Elisabeth Müller, Michael Gütschow · Acta Pharmaceutica Sinica B · 2024
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.
