An uniquely purified HCV NS3 protease and NS4A(21-34) peptide form a highly active serine protease complex in peptide hydrolysis.
The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4 degrees C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys-Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k(cat)/K(M)) of 205 and 196,000 M(-1) s(-1), respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease-NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k(cat)/K(M) of 5180 +/- 670 M(-1) s(-1)). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.
Sardana
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Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA. vinod_sardana@merck.com
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Zugay-Murphy
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Sardana
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Kuo
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D002623:Chemistry Techniques, Analytical / Q000379:methods; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000378:metabolism*; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D011506:Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus