3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism
3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism
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DOI：
10.1021/bi036022q
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发表时间：
2004-04-20
期刊：
影响因子：
2.9
通讯作者：
Lai, LH
中科院分区：
文献类型：
--
作者：
Huang, CK;Wei, P;Lai, LH
SARS 3C样蛋白酶被认为是抗SARS药物设计的关键酶。缺乏合适的检测方法是酶动力学研究和大规模筛选SARS 3CL蛋白酶抑制剂的主要障碍。由于SARS 3CL蛋白酶属于半胱氨酸蛋白酶家族（CB家族中的C3家族），具有胰凝乳蛋白酶折叠，因此了解SARS 3CL蛋白酶的催化机制以确定蛋白水解是通过胰凝乳蛋白酶等一般碱催化机制还是木瓜蛋白酶等离子对机制进行是很重要的。建立了SARS 3CL蛋白酶的连续比色法，并应用于酶催化机理的研究。所提出的催化残基His 41和Cys 145被证实通过突变为Ala对催化是关键的，而Cys 145突变为Set导致活性降低40倍的活性酶。根据催化活性的pH依赖性，野生型酶中His 41和Cys 145的pK（a）估计为6.38和8.34，而C145 S突变体中His 41和Ser 145的pK（a）估计分别为6.15和9.09。C145 S突变体在D2 O中具有正常的同位素效应，即在D2 O中反应较慢，而野生型酶显示出反向同位素效应，这可能来自较低的活化焓。测定的活性位点残基的pK（a）值和C145 S突变体的活性与一般碱催化机制一致，不能用硫醇盐-咪唑鎓离子对模型解释。
SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be critical for catalysis by mutating to Ala, while the Cys145 to Set mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pK(a)'s for His41 and Cys145 in the wild-type enzyme were estimated to be 6.38 and 8.34, while the pK(a)'s for His41 and Ser145 in the C145S mutant were estimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D2O for general base catalysis, that is, reacts slower in D2O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pK(a) values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.