Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer

A serine protease domain contained within the viral NS3 protein is a key pl ayer in the maturational processing of the hepatitis C virus polyprotein an d a prime target for the development of antiviral drugs. In the present wor k, we describe a dansylated hexapeptide inhibitor of this enzyme. Active si te occupancy by this compound could be monitored following fluorescence res onance energy transfer between the dansyl fluorophore and protein tryptopha n residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-stea dy-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. W e show that enzyme-product inhibitor complex formation depends on the prese nce of an NS4A cofactor peptide, Equilibrium and pre-steady-state data supp ort an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex for mation, requiring cofactor complexation prior to inhibitor binding.