Acta Pharmacologica Sinica 2005 January; 26 (1): 99-106; doi: 10.1111/j.1745-7254.2005.00010.x

Acta Pharmacologica Sinica 2005 January; 26 (1): 99-106; doi: 10.1111/j.1745-7254.2005.00010.x

Original Article

Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique¹

Shuai CHEN², Li-li CHEN², Hai-bin LUO, Tao SUN,Jing CHEN, Fei YE, Jian-hua CAI, Jing-kang SHEN, Xu SHEN³, Hua-liang JIANG³

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 201203, China

Aim: To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CL^pro) 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 3CL^pro proteolytic activity.

Results: The kinetic parameters of the fluorogenic substrate have been determined as K_m=404 mmol·L^-1, k_cat=1.08 min^-1, and k_cat/K_m=2.7 mmol^-1·L·min^-1. SARS-CoV 3CL^pro showed substantial pH and temperature-triggered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CL^pro 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 3CL^pro. This FRET-based assay might supply an ideal approach for the exploration SARS-CoV 3CL^pro putative inhibitors.

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

¹ 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.

² Authors contributed equally to this work.
³ Correspondence to Prof. Xu SHEN, Prof Hua-liang JIANG.
Phn 86-21-5080-6600. Fax 86-21-5080-7088.
E-mail xshen@mail.shcnc.ac.cn, hljiang@mail.shcnc.ac.cn
Received 2004-05-31 Accepted 2004-09-01

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