Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase.
Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase.
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DOI：
10.1074/jbc.m310875200
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发表时间：
2004-01-16
期刊：
影响因子：
--
通讯作者：
Lai L
中科院分区：
文献类型：
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作者：
Fan K;Wei P;Feng Q;Chen S;Huang C;Ma L;Lai B;Pei J;Liu Y;Chen J;Lai L
严重急性呼吸综合征（SARS）冠状病毒3C样蛋白酶被认为是基于结构的抗SARS药物设计的关键靶点。为了了解酶的活性形式和底物特异性，我们克隆、表达和纯化了SARS 3C样蛋白酶。分析凝胶过滤显示蛋白质浓度为4 mg/ml的单体和二聚体的混合物，并且大部分单体浓度为0.2mg/ml，其对应于酶测定中使用的浓度。酶的比活性随酶浓度的降低而线性降低，表明只有二聚体形式是有活性的，并且二聚体界面可以用于针对SARS 3C样蛋白酶的基于结构的药物设计。通过使用高压液相色谱分析，显示SARS 3C样蛋白酶以不同的效率切割覆盖病毒多聚蛋白上的所有11个切割位点的11个肽。对应于两个自切割位点的两个肽是具有最高切割效率的两个肽，而在P2或P1′位置具有非典型残基的肽反应较慢。底物的P2位置似乎有利于大的疏水残基。肽底物的二级结构研究表明，具有更多β-片层样结构的底物倾向于快速反应。本研究为SARS病毒3C样蛋白酶的酶催化作用和底物特异性谱的研究提供了基础知识，为基于结构的SARS病毒及其他冠状病毒抑制剂的设计提供了参考。
The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1′ positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more β-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.