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. 2005 Jan;26(1):99–106. doi: 10.1111/j.1745-7254.2005.00010.x

Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

Shuai Chen 1,#, Li-li Chen 1,#, Hai-bin Luo 1, Tao Sun 1, Jing Chen 1, Fei Ye 1, Jian-hua Cai 1, Jing-kang Shen 1, Xu Shen 1,✉, Hua-liang Jiang 1,✉
PMCID: PMC7091904  PMID: 15659121

Abstract

Aim:

To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed mutants.

Methods:

Based on the fluorescence resonance energy transfer (FRET) principle using 5-[(2′-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CLpro proteolytic activity.

Results:

The kinetic parameters of the fluorogenic substrate have been determined as Km=404 μmol·L−1, kcat=1.08 min−1, and kcat/Km=2.7 mmol−1·L·min−1. SARS-CoV 3CLpro showed substantial pH and temperature-triggered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CLpro revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity.

Conclusion:

This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARS-CoV 3CLpro putative inhibitors.

Keywords: severe acute respiratory coronavirus, 3C-like protease, fluorescence resonance energy transfer, fluorogenic substrate, enzyme activity, site-directed mutagenesis

Footnotes

Project supported by the State Key Program of Basic Research of China (grants 2003-CB514125, 2003CB514124, 2002CB512807, 2002CB512802, 2002AA233011), Sino-European Project on SARS Diagnostics and Antivirals (Proposal/Contract No 003831), and the special programs of oppugning SARS from the Ministry of Science and Technology, Chinese Academy of Sciences, National Natural Science Foundation of China and Shanghai Science and Technology Commission.

Shuai Chen and Li-li Chen: Authors contributed equally to this work.

Contributor Information

Xu Shen, FAX: 86-21-5080-7088, Email: xshen@mail.shcnc.ac.cn.

Hua-liang Jiang, FAX: 86-21-5080-7088, Email: hljiang@mail.shcnc.ac.cn.

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