Article

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

Drug Discovery and Design Center, 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.
Acta Pharmacologica Sinica (impact factor: 1.95). 02/2005; 26(1):99-106. DOI:10.1111/j.1745-7254.2005.00010.x pp.99-106
Source: PubMed

ABSTRACT To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CL(pro)) and its four site-directed mutants.
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.
The kinetic parameters of the fluorogenic substrate have been determined as Km=404 micromol.L(-1), kcat=1.08 min(-1), and kcat/Km=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.
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.

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Keywords

5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid
 
complete loss
 
exploration SARS-CoV 3CL(pro)
 
fluorescence resonance energy transfer
 
four site-directed mutants
 
FRET-based assay
 
kinetic parameters
 
present work
 
severe acute respiratory syndrome
 
substantial pH
 
temperature-triggered activity switches
 
valuable information