title: Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides.
abstract: We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k(cat)) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min(-1), respectively, and the values for the corresponding Michaelis-Menten constants (Km) were 280, 160, and 16 microM, providing catalytic efficiency values (k(cat)/Km) of 92, 1,130, and 8,300 M(-1) s(-1). An alanine-scanning study for an NS5A/5B substrate (P6P4') revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position.
authors.affiliation: Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. rumin.zhang@spcorp.com
authors.forename: R
authors.initials: R
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Durkin
authors.forename: W T
authors.initials: WT
authors.lastname: Windsor
authors.forename: C
authors.initials: C
authors.lastname: McNemar
authors.forename: L
authors.initials: L
authors.lastname: Ramanathan
authors.forename: H V
authors.initials: HV
authors.lastname: Le
mesh_terms: D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D010455:Peptides; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases