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

• DOI: 10.1016/j.apsb.2024.03.001
• Publication Date: 2024-03-06T16:49:19Z
• Authors: Rabea Voget; Julian Breidenbach; Tobias Claff; Alexandra Hingst; Katharina Sylvester; Christian Steinebach; Lan Phuong Vu; Renato H. Weiße; Ulrike Bartz; Norbert Sträter; Christa E. Müller; Michael Gütschow
• Keywords: Enzyme Kinetics; 2019-20 coronavirus outbreak

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