首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer
Authors:Fattori D  Urbani A  Brunetti M  Ingenito R  Pessi A  Prendergast K  Narjes F  Matassa V G  De Francesco R  Steinkühler C
Institution:Istituto di Ricerche di Biologia Molecolare "P. Angeletti" Via Pontina Km 30,600, Pomezia, 00040 Rome, Italy.
Abstract:A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.
Keywords:
本文献已被 PubMed 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号