Characterization of ATPase activity of a hepatitis C virus NS3 helicase domain, and analysis involving mercuric reagents.
The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) exhibits RNA-dependent NTPase/helicase activity. This enzyme is considered to be involved in viral replication and is expected to be one of the target molecules of anti-HCV drugs. In a search for NTPase inhibitors specific to HCV, we expressed and purified the truncated NS3 NTPase/helicase domain. Here, we report the characterization of its RNA-dependent ATPase activity. This enzyme preferred Mg(2+) and the optimal pH was 7.0. We further investigated the effects of heavy metal ions on the ATPase activity. The mercuric ion inhibited it significantly, the 50% inhibitory concentration being 49 nM. The fact that the inhibitory profile was competitive and that this inhibition was blocked in the presence of a large excess of cysteine or dithiothreitol, suggested that a cysteine residue in the DECH box was the main target site of mercury.
Kyono
Kiyoshi
K
Medicinal Chemistry Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505. k-kyono@tanabe.co.jp
Miyashiro
Masahiko
M
Taguchi
Ikuhiko
I
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001667:Binding, Competitive; D002412:Cations; D003553:Cystine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008628:Mercury / Q000494:pharmacology*; D008670:Metals / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D002412:Cations; D003553:Cystine; D007477:Ions; D008274:Magnesium; D008628:Mercury; D008670:Metals; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases