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J Biol Chem, Vol. 273, Issue 30, 18906-18914, July 24, 1998

Product Release Is the Major Contributor to k_cat for the Hepatitis C Virus Helicase-catalyzed Strand Separation of Short Duplex DNA

From Glaxo Wellcome, Research Triangle Park, North Carolina 27709

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). k_cat for this reaction was 0.12 s¹. 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 (~2) were disrupted concertedly. The maximal value of the rate constant (k_eff) describing the late phase of the reaction (strand separation) was 0.5 s¹, which was 4-fold greater than k_cat. Release of HF31 from E·HF31 in the presence of ATP (0.21 s¹) was the major contributor to k_cat. 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.

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