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

A titrant for the SARS-CoV-2 main protease (M ^pro ) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active M ^pro 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 M ^pro , 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 M ^pro -catalyzed cleavage of these substrates were determined.
Competing Interests: The authors declare no conflicts of interest.
(© 2024 The Authors.)