Two hydrophobic residues, W501 and V432, in the nucleic acid (NA) binding pocket of the HCV helicase domain (E) were mutagenized in an effort to investigate contributions of these residues to substrate affinities and to enzymatic activities. The affinities of wild-type [hE(wt)] and mutant enzymes [hE(W501F), hE(W501A), and hE(V432A)] for NA and ATP were determined by monitoring changes in the intrinsic protein fluorescence, in the fluorescence of fluorescently tagged nucleic acid, and in the enzymatic activity. The steady-state kinetic parameters of the mutant enzymes for ATP hydrolysis (at saturating concentrations of NA) were similar to those of hE(wt). hE(W501F), hE(W501A), and hE(V432A) had strand-separating activities that were 136%, 3.8%, and 3.1% of that of hE(wt). The processivities of hE(W501F), hE(W501A), and hE(V432A) were reduced relative to that of hE(wt). The reduced processivities of hE(W501F) and hE(W501A) were primarily due to an increase in the rate of dissociation of E. ATP from E.ATP.NA. The reduced processivity of hE(V432A) was primarily due to a reduction in the intrinsic forward rate constant for strand separation. This result suggested that V432 may constitute part of the forward "stepping" motor of E. hE(W501A) and hE(V432A) did not display a dominant negative phenotype in a steady-state helicase assay with hE(wt). hE(wt) stored in the presence of beta-mercaptoethanol was covalently modified at three cysteinyl residues. The biological significance of the potential reactivity of these cysteinyl residues on hE(wt) is unknown.