Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique
文献类型：期刊论文
| 作者 | Chen, S; Chen, LL ; Luo, HB; Sun, T; Chen, J; Ye, F; Cai, JH; Shen, JK; Shen, X; Jiang, HL
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| 刊名 | ACTA PHARMACOLOGICA SINICA
 |
| 出版日期 | 2005-01 |
| 卷号 | 26期号:1页码:99-106 |
| 关键词 | severe acute respiratory coronavirus 3C-like protease fluorescence resonance energy transfer fluorogenic substrate enzyme activity site-directed mutagenesis |
| ISSN号 | 1671-4083 |
| DOI | 10.1117/j.1745-7254.2005.00010.x |
| 文献子类 | Article |
| 英文摘要 | 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 wag 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 mumolcircleL(-1), k(cat)=1.08 min(-1), and k(cat)/K(m)=2.7 mmol(-1)circleLcirclemin(-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 His(41), Cys(145), and His(163) resulted in complete loss of enzymatic activity, while replacement of Met(162) 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 3CLP(pro) putative inhibitors. |
| WOS关键词 | ACUTE RESPIRATORY SYNDROME ; VIRUS-ENCODED PROTEINASES ; FLUOROGENIC SUBSTRATE ; MUTATIONAL ANALYSIS ; RNA-POLYMERASE ; MAIN PROTEASE ; SEQUENCE ; PURIFICATION ; POLYPROTEIN ; INHIBITOR |
| WOS研究方向 | Chemistry ; Pharmacology & Pharmacy |
| 语种 | 英语 |
| CSCD记录号 | CSCD:1874634 |
| WOS记录号 | WOS:000226426600015 |
| 出版者 | SHANGHAI INST MATERIA MEDICA |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/273947]  |
| 专题 | 药理学第三研究室 中科院受体结构与功能重点实验室 新药研究国家重点实验室 药物发现与设计中心 |
| 通讯作者 | Chen, S |
| 作者单位 | Chinese Acad Sci, Grad Sch,Shanghai Inst Mat Med, Shanghai Inst Biol Sci, State Key Lab Drug Res,Drug Discovery & Design Ct, Shanghai 201203, Peoples R China |
| 推荐引用方式 GB/T 7714 | Chen, S,Chen, LL,Luo, HB,et al. Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique[J]. ACTA PHARMACOLOGICA SINICA,2005,26(1):99-106. |
| APA | Chen, S.,Chen, LL.,Luo, HB.,Sun, T.,Chen, J.,...&Jiang, HL.(2005).Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique.ACTA PHARMACOLOGICA SINICA,26(1),99-106. |
| MLA | Chen, S,et al."Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique".ACTA PHARMACOLOGICA SINICA 26.1(2005):99-106. |
入库方式： OAI收割
来源：上海药物研究所
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