A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus. The first cycle of interaction of ATP with the enzyme is unique.
Hepatitis C virus (HCV) helicase (E) formed spectrofluorometrically detectable complexes with a 16-mer and HF16 (a 16-mer with 5'-hexachlorofluoresceinyl moiety). The interaction of helicase with these effectors was investigated by kinetic techniques to determine if the complexes were kinetically competent for ATP hydrolysis. kcat values with the 16-mer and HF16 were 2.7 and 36 s-1, respectively. The maximal value of the rate constant for the approach of an intermediate to the steady-state level has to be at least 4-fold greater than kcat for it to be kinetically competent. This value was 1.2 s-1 with HF16 and "E.ATP" and was 1.82 s-1 with ATP and E.HF16. These values were too small for formation of these intermediates to be kinetically competent in ATP hydrolysis. Dissociation of "E.HF16. ATP" (0.34 s-1) was also too slow to contribute significantly to catalysis. Furthermore, the Km of E.HF16 for ATP (3 mircoM) was significantly less than the Km for ATP hydrolysis at a saturating concentration of HF16 (320 microM). HCV helicase has two nucleotide-binding sites per monomer. If the fluorescence changes observed were associated with structure changes preceding steady-state catalysis (isomerization), pre-steady-state data could be reconciled with the turnover data. Data for the 16-mer yielded similar conclusions.
Porter
D J
DJ
Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry*; D005452:Fluoresceins / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology*; D007536:Isomerism; D007700:Kinetics; D009841:Oligonucleotides / Q000494:pharmacology*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; D005452:Fluoresceins; D009841:Oligonucleotides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases