3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis   by a general base mechanism

doi:10.1021/bi036022q

CORC > 北京大学 > 化学与分子工程学院

	3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism
	Huang, CK ; Wei, P ; Fan, KQ ; Liu, Y ; Lai, LH
刊名	biochemistry us
	2004
关键词	STRUCTURE-BASED DESIGN 3C PROTEASE INHIBITORS ACUTE RESPIRATORY SYNDROME IMIDAZOLIUM ION-PAIR BIOLOGICAL EVALUATION MAIN PROTEINASE ACTIVE-SITE 2-PYRIDONE-CONTAINING PEPTIDOMIMETICS PURIFICATION PAPAIN
DOI	10.1021/bi036022q
英文摘要	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.; http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000220812900018&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701 ; Biochemistry & Molecular Biology; SCI(E); EI; PubMed; 73; ARTICLE; 15; 4568-4574; 43
语种	英语
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/199608]
专题	化学与分子工程学院
推荐引用方式 GB/T 7714	Huang, CK,Wei, P,Fan, KQ,et al. 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism[J]. biochemistry us,2004.
APA	Huang, CK,Wei, P,Fan, KQ,Liu, Y,&Lai, LH.(2004).3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.biochemistry us.
MLA	Huang, CK,et al."3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism".biochemistry us (2004).