Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.
Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.
Porter
D J
DJ
Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA.
Short
S A
SA
Hanlon
M H
MH
Preugschat
F
F
Wilson
J E
JE
Willard
D H
DH
Consler
T G
TG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D004265:DNA Helicases / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011817:Rabbits; D014461:Ultracentrifugation; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000255:Adenosine Triphosphate; D004265:DNA Helicases; D004279:DNA, Viral; D012316:RNA Nucleotidyltransferases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases