3C-like Proteinase from SARS Coronavirus Catalyzes Substrate Hydrolysis by a General Base Mechanism

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• 作者：Changkang Huang ; Ping Wei ; Keqiang Fan ; Ying Liu ; and Luhua Lai
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：April 20, 2004
• 年：2004
• 卷：43
• 期：15
• 页码：4568 - 4574
• 全文大小：65K
• 年卷期：v.43,no.15(April 20, 2004)
• ISSN：1520-4995

文摘

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 inhibitorscreen 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 mechanismof SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysismechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuouscolorimetric 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 mutatingto 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 enzymewere estimated to be 6.38 and 8.34, while the pK_a's for His41 and Ser145 in the C145S mutant wereestimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D₂O forgeneral base catalysis, that is, reacts slower in D₂O, while the wild-type enzyme shows an inverse isotopeeffect which may come from the lower activation enthalpy. The pK_a values measured for the active siteresidues and the activity of the C145S mutant are consistent with a general base catalysis mechanism andcannot be explained by a thiolate-imidazolium ion pair model.