Huang Changkang, Wei Ping, Fan Keqiang, Liu Ying, Lai Luhua
State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Biochemistry. 2004 Apr 20;43(15):4568-74. doi: 10.1021/bi036022q.
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 Ser 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 D(2)O for general base catalysis, that is, reacts slower in D(2)O, 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.
严重急性呼吸综合征（SARS）3C样蛋白酶已被认为是针对SARS进行药物设计的关键酶。缺乏合适的检测方法一直是酶动力学研究以及针对SARS 3CL蛋白酶进行大规模抑制剂筛选的主要障碍。由于SARS 3CL蛋白酶属于具有胰凝乳蛋白酶折叠的半胱氨酸蛋白酶家族（CB族中的C3家族），了解SARS 3CL蛋白酶的催化机制对于确定蛋白水解是通过类似胰凝乳蛋白酶的一般碱催化机制还是类似木瓜蛋白酶的离子对机制进行至关重要。我们建立了一种针对SARS 3CL蛋白酶的连续比色测定法，并将其应用于研究酶的催化机制。通过突变为丙氨酸，证实了所提出的催化残基His41和Cys145对催化至关重要，而Cys145突变为Ser产生了一种活性降低40倍的活性酶。根据催化活性对pH的依赖性，野生型酶中His41和Cys145的pK(a)值估计分别为6.38和8.34，而C145S突变体中His41和Ser145的pK(a)值分别估计为6.15和9.09。C145S突变体在D(2)O中对于一般碱催化具有正常的同位素效应，即在D(2)O中反应较慢，而野生型酶显示出反向同位素效应，这可能源于较低的活化焓。活性位点残基测得的pK(a)值和C145S突变体的活性与一般碱催化机制一致，无法用硫醇盐-咪唑鎓离子对模型来解释。
