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

作　　者：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 

机构地区：[1]Pharmaceutical Institute,Pharmaceutical&Medicinal Chemistry,University of Bonn,Bonn 53121,Germany [2]Institute of Bioanalytical Chemistry,Center for Biotechnology and Biomedicine,Leipzig University,Leipzig 04103,Germany [3]Department of Natural Sciences,University of Applied Sciences Bonn-Rhein-Sieg,Rheinbach 53359,Germany

出　　处：《Acta Pharmaceutica Sinica B》2024年第5期2349-2357,共9页药学学报（英文版）

基　　金：The authors acknowledge support by Dr.Carina Lemke and Marion Schneider.Christa E.Müller and Michael Gütschow were supported by the Volkswagen Foundation(9A894);Rabea Voget,Christian Steinebach,Christa E.Müller and Michael Gütschow by the German Research Foundation(RTG 2873);Norbert Sträter by the Volkswagen Foundation(9A850).We acknowledge DESY(Hamburg,Germany),a member of the Helmholtz Association HGF,and the EMBL for the provision of experimental facilities at synchrotron beamlines P13 and P14 and the MX Laboratory at the Helmholtz Zentrum Berlin(BESSY II)for beam time.We would like to thank Selina Storm for assistance in using the EMBL beamlines.

摘　　要：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.

关 键 词：COVID-19 SARS-CoV-2 Main protease Peptide nitriles Fluorogenic substrates Active-site titration X-ray crystallography Inner filter effect 

分 类 号：R917[医药卫生—药物分析学]