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

David J. T. PorterDagger , Steven A. Short, Mary H. Hanlon, Frank Preugschat, Jeanne E. Wilson, Derril H. Willard Jr., and Thomas G. Consler

From Glaxo Wellcome, Research Triangle Park, North Carolina 27709

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (~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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Helicases are ubiquitous enzymes required for cellular repair, recombination, and replication (1, 2). Even though helicase activity was identified and the associated protein was purified over 20 years ago (3), the kinetic and chemical mechanism for this class of enzymes is unknown. Lohman and co-workers (4-13) have initiated an extensive effort to elucidate the mechanism of action of Escherichia coli Rep helicase. They have proposed that the catalytically active species is a dimeric form of the enzyme stabilized by DNA (2, 4, 12). The two DNA-binding sites in the catalytically active dimer unwind defined lengths of the DNA duplex by alternatively binding duplex and single-stranded DNA during the catalytic cycle (2, 6, 7, 13). Recently, Ali and Lohman (14) reported that E. coli helicase II catalyzes the unwinding of defined duplexes with a step size of 4 to 5 base pairs per catalytic cycle. These results supported an active mechanism for separating double-stranded DNA in which the step size is relatively small.

Our interest in helicases is based on the observation that the HCV1 genome encodes a unique helicase within the NS3 protein. Because approximately 1% of the population is infected with HCV and available therapies are effective for only a small subpopulation of these patients, an urgent medical need exists for an effective anti-HCV agent (15-17). Consequently, HCV helicase is an attractive target for development of an antiviral agent for HCV.

The NS3 protein of HCV has at least two enzymatic activities necessary for viral replication. The N-terminal 20 kDa of NS3 is a serine proteinase that cleaves the HCV-encoded polyprotein at four specific positions (18). The C-terminal 50 kDa of NS3 has NTPase (19) and RNA helicase activity (20). We have initiated a program to characterize the kinetic and chemical mechanism of action for the HCV helicase domain. The kinetic mechanism of the ATPase activity associated with this protein isolated from HCV genotype 1b, which is a major subtype found in both Japanese and American populations (21), has been characterized in detail (22). Herein, we extend our understanding of the kinetic mechanism of the helicase activity of this protein by analysis of the strand separation reaction with fluorescently tagged duplex oligomers of defined sequences. The results with HCV helicase were consistent with a passive kinetic mechanism for DNA strand separation in which dissociation of single-stranded DNA was the major contributor to kcat.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Rabbit muscle pyruvate kinase, rabbit muscle lactate dehydrogenase, NADH, phosphoenolpyruvate, adenosine 5'-triphosphate, AMPCPP, ATPgamma S, ADPPNP, MOPS, Tris, ammonium sulfate, and dextran sulfate (Mr = 5000) were from Sigma. ATP, poly(rU), and single-stranded DNA cellulose resin were from Amersham Pharmacia Biotech. All oligomers of defined sequence were purchased from Oligo Therapeutics, Inc., and were purified by electrophoresis through a 6 M urea, 20% polyacrylamide gel.

Purification of the HCV Helicase-- The enzyme was purified as described previously except that a single-stranded DNA cellulose column was substituted for the Bio-Gel P-100 column in the previous purification protocol (22). In summary, the enzymatic activity from the Mono Q purification step (22) was absorbed onto a column of the single-stranded DNA-cellulose resin (10 g) that was equilibrated in the purification buffer. The flow rate was 1 ml/min. After washing the resin with 10 ml of buffer containing 0.15 M NaCl, the protein was eluted from the resin with buffer containing 1.0 M NaCl. The purified enzyme was stored at -20 °C for several months without loss of catalytic activity.

Assay of HCV Helicase Activities-- The ATPase activity associated with HCV helicase was monitored by the ADP-dependent oxidation of NADH in the lactate dehydrogenase and pyruvate kinase-coupled reaction as described previously (22).

Several continuous read-out fluorescence-based assays have been recently developed for the study of DNA strand separation catalyzed by helicases (10, 13, 23-27). For the HCV helicase reaction, we chose the fluorescence resonance energy assay developed by Bjornson et al. (10). The substrate was a double-stranded DNA of fluorescein- and hexachlorofluorescein-tagged oligomers (10). The fluorescence of the fluorescein moiety (lambda ex = 492 nm and lambda em = 522 nm) in the double-stranded DNA was quenched by fluorescence resonance energy transfer to the hexachlorofluorescein moiety (10). Separation of the fluorescein and hexachlorofluorescein moieties results in an increase in fluorescence of the fluorescein moiety (10). For our studies, a 21-mer and a complementary 31-mer were modified with fluorescein and hexachlorofluorescein on the 3' end (F21) and 5' end (HF31), respectively. The fluorescence quenching we observed upon formation of F21:HF31 was approximately 2-fold greater than that observed by Bjornson et al. (10) for the formation of an analogously tagged duplex DNA. The fluorescence changes associated with strand separation of F21:HF31 by sub-stoichiometric concentrations of enzyme were proportional to the fractional strand separation of F21:HF31 determined by a gel-based assay (data not shown).

Kinetic and Titration Data Analyses-- Equation 1, which describes a random rapid equilibrium or steady-state ordered mechanism (28), was fitted to the steady-state data to determine the value of kcat for strand separation of F21:HF31.
<FR><NU>v</NU><DE>E<SUB>t</SUB></DE></FR>=<FR><NU>k<SUB><UP>cat</UP></SUB>[<UP>ATP</UP>][<UP>F21:HF31</UP>]</NU><DE><AR><R><C>[<UP>ATP</UP>][<UP>F21:HF31</UP>]+K<SUB><UP>ATP,F21:HF31</UP></SUB>[<UP>ATP</UP>]</C></R><R><C>+K<SUB><UP>F21:HF31,ATP</UP></SUB>[<UP>F21:HF31</UP>]+K<SUB><UP>ATP</UP></SUB>K<SUB><UP>ATP,F21:HF31</UP></SUB></C></R></AR></DE></FR> (Eq. 1)
KATP, F21:HF31 is the Km of the enzyme for F21:HF31 extrapolated to infinite ATP concentration; KF21:HF31,ATP is the Km of the enzyme for ATP extrapolated to infinite F21:HF31 concentration; and KATP is the Kd of the enzyme for ATP in the absence of F21:HF31. The time courses for changes in the intrinsic protein fluorescence resulting from binding of DNA to helicase were described by either a single (n = 1) or double (n = 2) exponential function (Equation 2).
F(t)=F<SUB>0</SUB>+<LIM><OP>∑</OP><LL>i<UP>=</UP>1</LL><UL>n</UL></LIM> F<SUB>i</SUB>e<SUP>(<UP>−</UP>k<SUB>i</SUB>·t)</SUP> (Eq. 2)
The dependence of the kobs values on DNA concentration ([L]) was described by either a two-step mechanism or a one-step mechanism (Equation 3 or 4).
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>−</UP>1</SUB>+k<SUB>1</SUB> <FR><NU>[<UP>L</UP>]</NU><DE>(K+[<UP>L</UP>])</DE></FR> (Eq. 3)
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>−</UP>1</SUB>+k<SUB>s</SUB>[<UP>L</UP>] (Eq. 4)

Equations 5 and 6 were fitted to the biphasic fluorescence changes associated with HCV-catalyzed strand separation of F21:HF31, 21:HF31, and 42:HF52 in half-reactions or partial turnover experiments. The early phase was a linear change in fluorescence from an initial value (c) to a final value (d) over a time interval (a) (Equation 5). The late phase was an exponential change in fluorescence from an initial value (d) to the final fluorescence (e) (Equation 6).
F(t)=c+t<FENCE><FR><NU>(d−c)</NU><DE>a</DE></FR></FENCE>  t<a (Eq. 5)
F(t)=e+(d−e)<UP>exp</UP><SUP><UP>−</UP>b(t<UP>−</UP>a)</SUP>  t>a (Eq. 6)

Sedimentation Velocity Centrifugation-- Sedimentation velocity analytical ultracentrifugation was performed with a Beckman (Fullerton, CA) XL-A centrifuge with two-channel 12-mm charcoal-filled epon centerpieces. Scans were taken at 280 nm for HCV helicase alone or at 492 nm for experiments with 3' fluorescein-tagged DNA (I-F) at 10-min intervals over a 5-h period. Centrifugation was at 40,000 rpm at 4 °C, except in the case of I-F alone, which was performed at 60,000 rpm. Each experimental solution (200 µl) was centrifuged against 250 µl of the equivalent blank. The apparent sedimentation coefficient, Sapp, was calculated from the apparent differential sedimentation coefficient distribution, using a software analysis package created by Stafford (29) that was obtained through the National Analytical Ultracentrifugation Facility at the University of Connecticut (Storrs, CT).

Sedimentation Equilibrium Centrifugation-- Sedimentation equilibrium analytical ultracentrifugation was performed as described above except that six-sectored cells were utilized. Scans were taken at 280 nm at 1-h intervals throughout the run. Runs were performed at 20,000, 25,000, and 30,000 rpm at 4 °C. Equilibrium was achieved after approximately 20 h. Each sample (80-100 µl) was centrifuged against 120 µl of the equivalent blank. Solvent density was determined empirically at 4 °C using a Mettler (Highstown, NJ) DA-110 density/specific gravity meter calibrated against water. The partial specific volume of each protein, <A><AC>&ngr;</AC><AC>¯</AC></A>, was calculated using the method of Cohn and Edsall (30). Adjustments for temperature were made using the appropriate equation that has been modified to use <A><AC>&ngr;</AC><AC>¯</AC></A> values derived for each amino acid at 25 °C (31). The partial specific volume of HCV helicase was calculated to be 0.726 ml/g at 4 °C. The value of <A><AC>&ngr;</AC><AC>¯</AC></A> for I was 0.55 ml/g. Data were collected as radial distance versus absorbance. The data were analyzed by an adaptation of the Beckman/Microcal Origin nonlinear regression software package using multiple iterations of the Marquardt-Levenberg algorithm for parameter estimation. Multiple models were employed to determine the most accurate description of the macromolecular solution state.
A<SUB>r</SUB>=A<SUB>01</SUB>e<SUP><FENCE><FR><NU>(1<UP>−</UP>&ngr;<SUB>1</SUB>&rgr;)&ohgr;<SUP>2</SUP></NU><DE>2RT</DE></FR>M<SUB>1</SUB>(r<SUP>2</SUP><UP>−</UP>r<SUP>2</SUP><SUB>0</SUB>)</FENCE></SUP>+A<SUB>02</SUB>e<SUP><FENCE><FR><NU>(1<UP>−</UP>&ngr;<SUB>2</SUB>&rgr;)&ohgr;<SUP>2</SUP></NU><DE>2RT</DE></FR>M<SUB>2</SUB>(r<SUP>2</SUP><UP>−</UP>r<SUP>2</SUP><SUB>0</SUB>)</FENCE></SUP> (Eq. 7)
+<FENCE>A<SUP>n<SUB>1</SUB></SUP><SUB>01</SUB>+A<SUP>n<SUB>2</SUB></SUP><SUB>02</SUB></FENCE>Ke<SUP><FENCE><FENCE>n<SUB>1</SUB><FR><NU>(1<UP>−</UP>&ngr;<SUB>1</SUB>&rgr;)&ohgr;<SUP>2</SUP></NU><DE>2RT</DE></FR>M<SUB>1</SUB><UP>+</UP>n<SUB>2</SUB><FR><NU>(1<UP>−</UP>&ngr;<SUB>2</SUB>&rgr;&ohgr;<SUP>2</SUP></NU><DE>2RT</DE></FR>M<SUB>2</SUB></FENCE>(r<SUP>2</SUP><UP>−</UP>r<SUP>2</SUP><SUB>0</SUB>)</FENCE></SUP>+E
The sedimentation of a single, ideal species was described by the first term on the right-hand side of Equation 7. Ar was the absorbance at radial position r. A01 was the absorbance of the single species monomer at r0 (the reference radial position at the top of the gradient). <A><AC>&ngr;</AC><AC>¯</AC></A>1 was the partial specific volume; rho  was the solvent density; omega  was the angular velocity; R was the gas constant; T was the temperature in degrees Kelvin; and M1 was the molecular mass. The base-line offset was E. This analysis yielded an approximate value for the mass average molecular mass of the sedimenting species in the system. This single ideal species model was sufficient to describe the sedimentation of helicase or DNA alone. The description of the sedimentation of helicase in the presence of DNA required all the terms of Equation 7, which included terms for each of the individual sedimenting monomeric species, the hetero-complex species, and a base-line offset. The variables were defined as for the single, ideal species model. The variables subscripted with 1 and 2 refer to parameters for helicase and DNA, respectively. In addition, n1 and n2 referred to the number of molecules of helicase and DNA, respectively, that were in the hetero-complex.

General Methods-- The standard buffer was 0.05 M MOPS (K+), 3.5 mM MgCl2 at pH 7.0. The standard temperature was 25 °C. Duplex DNA was made from stoichiometric concentrations of single-stranded DNA by heating the solution to 90 °C for 3 min and then cooling to room temperature over several hours. Fluorescence spectra were recorded on a Kontron SFM 25 spectrofluorometer. Intrinsic protein fluorescence data (lambda ex = 280-300 nm and lambda em = 340 nm) were corrected for filter effects. F21 was monitored with lambda ex = 492 nm and lambda em = 522 nm; HF31 was monitored with lambda ex = 510 nm and lambda em = 552 nm with a 2-mm slit. Absorbance and conventional kinetic data were obtained from a UVIKON 860 spectrophotometer. Rapid reactions were monitored on an Applied Photophysics SX.17MV Spectrophotometer (Leatherhead, UK). Entrance and exit slits were 10 mm in the absorbance mode and 2 mm in the fluorescence mode. The fluorescence of F21 and HF31 was monitored on the stopped-flow spectrofluorometer with lambda ex = 492 nm and lambda ex >530. The intrinsic protein fluorescence was monitored with lambda ex between 280 and 290 nm and lambda ex >305 or >320 nm. Stopped-flow tracings were an average of 4-6 experiments. The appropriate equations were fitted to the data by nonlinear least squares using SigmaPlot from Jandel Scientific (Corte Madera, CA).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Nucleotide Triphosphates and DNA Did Not Induce Oligomerization of HCV Helicase-- HCV helicase is isolated as a monomeric protein (22). Many helicases that are monomeric in the absence of substrates undergo substrate-induced oligomerization to the catalytically active form (32, 33). This frequently results in a nonlinear dependence of the rate of the reaction on enzyme concentration. For instance, the ATPase activity of E. coli Rep helicase, which undergoes substrate-induced dimerization, is a nonlinear function of enzyme concentration (2, 12). The ATPase activity of HCV helicase, however, was linearly dependent on helicase concentration between 0 to 50 nM enzyme with 1000 µM ATP and 600 µM poly(rU) (monomer). Similarly, the DNA unwinding activity was linearly dependent on helicase concentration over a range of 0 to 25 nM enzyme with 2000 µM ATP and 17 nM F21:HF31 as substrates. These results suggested that either the oligomerization of HCV helicase did not change over this range of enzyme concentrations or changes in oligomerization of the enzyme did not affect catalysis.

The oligomerization of HCV helicase in the presence of substrate analogues was investigated further by sedimentation equilibrium centrifugation. A duplex DNA with a stem-loop structure (GGC CTA AGC GTA TCG CTT AGG CCG AGT CAG G, I) was chosen for these studies because 1) it bound the helicase tightly with a 1:1 stoichiometry, 2) it mimicked double-stranded DNA and RNA that HCV helicase normally unwinds (34, 35), and 3) if unwound by the enzyme, it would reanneal rapidly. Sedimentation equilibrium data for HCV helicase alone or I alone (4 to 20 µM) were described by the first term of Equation 7 that described sedimentation of a single ideal species. The fitted values for the molecular mass of enzyme and I were 49,000 ± 1000 and 12,000 ± 1000 Da, respectively, which were within error of that expected for the respective monomeric macromolecules.

Solutions of mixtures of helicase and I exhibited more complicated sedimentation behavior. A model that includes one hetero-associating species was found to adequately fit the experimental results (Equation 7). Goodness of the fit was judged by several criteria as follows: 1) the value of the reduced sums of the squares of the residuals, 2) a random distribution pattern of the residuals to the fit, and 3) the appropriate nature of the model to the system being studied. Fig. 1 represents an example of the best fit to this model. The random distribution of residuals is shown above the data. The apparent dissociation constant for the 1:1 hetero-complex was less than 1 µM.2 Species distribution analysis using this association constant indicated that greater than 70% of the macromolecules were in this hetero-complex under these experimental conditions. Attempts to fit models to the data having hetero-species with other than 1:1 stoichiometry resulted in a less random distribution of residuals and values for association constants that translated to a very small (less than 5%) hetero-species population.


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Fig. 1.   Equilibrium analytical ultracentrifugation of HCV helicase·I. A mixture of 10 µM helicase and 10 µM I were centrifuged at 20,000 RPM and 4 °C in the standard buffer. Data are represented as absorbance at 280 nm versus radial position. Sedimentation gradients were analyzed by nonlinear curve fitting using equations describing a heterogeneous molecular association model, as described under "Experimental Procedures." The solid line through the data points was the best fit to the data for a 1:1 complex between helicase and I. Residuals to the best fit to the data are shown above the data.

Because the HVC helicase has significant ATPase activity, it was not experimentally feasible to determine the effect of ATP on the oligomerization of the enzyme by sedimentation equilibrium centrifugation. Consequently, the effects of I and ATP analogues on the oligomerization of HCV helicase were investigated by sedimentation velocity centrifugation. In these experiments, I tagged on the 3' end with a fluorescein moiety (I-F) allowed sedimentation of E·I-F to be monitored at wavelengths (lambda max = 492 nm) without influence from nucleotides (lambda max = 260 nm). The Sapp values for E·I-F in the presence or absence of ATP analogues were similar (Table I). If the substrates induced dimerization of the helicase, the sedimentation coefficient would have been expected to increase by 50% assuming helicase was accurately described by a spherical molecule (32). Thus, the sedimentation velocity and equilibrium data suggested that I·F or the combination of I·F and ATP analogues did not affect oligomerization of this protein.

                              
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Table I
Effect of ATP analogues on the sedimentation velocity of E·I-F at 4 °C

Steady-state Kinetics for Unwinding of HF31:F21 by HCV Helicase and ATP-- Initial velocity data for unwinding F21:HF31 by HCV helicase ([E] <<  [F21:HF31]) and ATP were monitored as a fluorescence increase associated with enzymatic separation of the duplex (Fig. 2). These data were analyzed by Equation 1. The value of kcat was 0.123 ± 0.009 s-1. The values of the other parameters were KATP, F21:HF31 = 0.030 ± 0.006 µM, KF21:HF31, ATP = 360 ± 90 µM, and KATP = 1600 ± 400 µM.


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Fig. 2.   Steady-state velocities for unwinding of F21:HF31 by helicase as a function of ATP and F21:HF31 concentrations. Strand separation of F21:HF 31 was monitored as described under "Experimental Procedures." The reactions were initiated with 1.1 nM helicase. The slope of the time course for the fluorescence change was calculated over approximately 100 s. Equation 1 was fitted to the data to give values for kcat = 0.124 s-1, KATP, F21:HF31 = 0.030 µM, KF21:HF31,ATP = 360 µM, and KATP = 1600 µM. The solid lines were calculated with these fitted values and Equation 1.

Reaction of E·ATP with F21:HF31 Monitoring Enzyme Fluorescence-- The reaction of E·ATP (because of the ATPase activity of the enzyme this species was a possible mixture of E·ATP, E·ADP·Pi, and E·ADP and E·Pi) with excess F21:HF31 and ATP (Fig. 3) resulted in a time-dependent decrease in intrinsic protein fluorescence resulting from formation of E·ATP·F21:HF31 (Equation 8). Because ATP and F21:HF31 were in excess of enzyme, the fluorescence changes were monitoring the approach of the system to the steady state.


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Fig. 3.   Kinetics for the approach to the steady-state starting with E·ATP and F21:HF31. The reaction of E·ATP with F21:HF31 was monitored by the change in intrinsic protein fluorescence (lambda ex = 280 nm and lambda em >305 nm) associated with the formation of E·ATP·F21:HF31 at large concentrations of ATP (inset). The time course for the reaction of 50 nM E·ATP with 400 nM F21:HF31 in the presence of 1000 µM ATP was first-order in enzyme concentration (inset). Equation 2 (i = 1) was fitted to these data to give a pseudo first-order rate constant of 2.4 s-1. The value of kobs for the reaction of 240 nM F21:HF31 was similar with 500 and 2000 µM ATP. The value of the bimolecular rate constant for association of E·ATP with F21:HF31 was 3.4 µM-1 s-1 (k1,ATP in Equation 9). The intercept value was 0.96 s-1.

The pseudo first-order rate constant for the approach to the steady-state (kobs) for the simplified scheme of Equation 8 was given by Equation 9. The values of kobs were linearly dependent on F21:HF31 concentration (Fig. 3) and similar for E·ATP formed with 500, 1000, and 2000 µM ATP (data not shown).
k<SUB><UP>obs</UP></SUB>=k<SUB>1,<UP>ATP</UP></SUB>[<UP>F21:HF31</UP>]+k<SUB><UP>−1,ATP</UP></SUB>+k<SUB><UP>cat</UP></SUB> (Eq. 9)
The values calculated for k1,ATP and the sum of kcat and k-1,ATP were 3.4 ± 0.1 µM-1 s-1 and 0.96 ± 0.09 s-1, respectively (Equation 9). Thus, the calculated value of k-1,ATP was 0.84 s-1 (kcat = 0.12 s-1). The value of k1,ATP was similar to the value of kcat/kATP, F21:HF31 (4.1 µM-1 s-1) from steady-state data indicating that the pre-steady-state data for the reaction of E·ATP with F21:HF31 was measuring an event associated with catalysis.

Half-reaction of E·F21:HF31 with ATP: Processivity of E·F21:HF31-- The fraction of F21:HF31 in E·F21:HF31 that was separated into F21 and HF31 (separation of fluorescent probes) after addition of ATP in the presence or absence of excess single-stranded competitor was estimated from the increase in fluorescence of fluorescein (lambda ex = 492 nm and lambda em = 522 nm). E·F21:HF31 (50 nM) was formed from stoichiometric amounts of enzyme and F21:HF31 in the absence of ATP (Kd = 0.4 nM). In the absence of competitor (dU)18, the increase in fluorescence of F21 after addition of 2000 µM ATP was the result of total strand separation (inset Fig. 4 with addition of the mixture of ATP and (dU)18 at the time indicated by the arrow). In the presence of competitor (dU)18, only a fraction of F21:HF31 strands was separated. This result suggested that a significant fraction of F21:HF31 or partially unwound F21:HF31 in E·F21:HF31·ATP dissociated from the enzyme prior to strand separation (i.e. the processivity of the enzyme was low). The ratio of the fluorescence changes in the presence (dU)18 to that in the absence was dependent on ATP concentration. The maximal fractional strand separation extrapolated to infinite ATP concentration was 0.44 ± 0.03. The concentration of ATP that yielded 50% of the maximal fractional strand separation was 1300 ± 200 µM (Fig. 4). The fractional strand separation was not affected by enzyme concentration (200 nM enzyme, 50 nM F21:HF31), which suggested that multiple enzyme molecules were not binding to a single molecule of F21:HF31.


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Fig. 4.   Dependence of fractional strand separation of F21:HF31 in E·F21:HF31 on ATP concentration in the presence of competitor DNA. The formation of F21 from F21:HF31 in the presence of ATP and HCV helicase was monitored by the fluorescence increase at 522 nm (lambda ex = 492 nm). E·F21:HF31 was formed from 50 nM F21:HF31 and 58 nM E. The reaction was initiated by the simultaneous addition of selected concentrations of ATP and 4.0 µM (dU)18 to trap free enzyme as F21:HF31 or strand separated DNA was released from E·F21:HF31·ATP. An example of the time course for the reaction with 2000 µM ATP in the absence of (dU)18 (total strand separation) or in the presence of 4.0 µM (dU)18 (partial strand separation) is presented in the inset. At the time indicated by the arrow the mixture of ATP and (dU)18 was added simultaneously to E·F21:HF31. The fractional strand separation (ratio of the fluorescence change in the presence of (dU)18 to that in the absence of (dU)18) was depended on the ATP concentration. The maximal fractional strand separation in the presence of ATP and (dU)18 was 0.44 and the concentration of ATP yielding 50% of this value was 1300 µM.

Half-reaction of E·F21:HF31 with ATP: Kinetics of Strand Unwinding and Strand Separation-- The half-reaction of 50 nM E·F21:HF31 with 2000 µM ATP in the presence of 40 µM competitor DNA ((dU)18), which was added simultaneously with ATP to E·F21:HF31, resulted in a biphasic fluorescence increase (lambda ex = 492 nm and lambda em >530 nm, Fig. 5). Under these conditions E·F21:HF31 was converted to 30% enzyme-free F21, 30% enzyme-free HF31, and 70% F21:HF31. Equations 5 and 6 were fitted to these data with a = 0.78 ± 0.01 s, b = 0.26 ± 0.01 s-1, c = 0 (fixed), d = -0.001 ± 0.0002, and e = 1 (fixed). Because the observation wavelengths were monitoring fluorescence changes associated with formation of F21 and dissociation of E·HF31 (see below), the kinetics of the late phase of the reaction were complicated. Nonetheless, the time course for the reaction demonstrated a distinct lag (0.78 s) prior to formation of enzyme-free F21 and HF31. The early phase was attributed to unwinding of the duplex prior to complete strand separation. Because the fluorescent probes were not separated during this phase, the fluorescent change associated with this reaction was small. The late phase was attributed to strand separation. Because the fluorescent probes were separated in this phase, the fluorescent changes were large (see "Discussion").


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Fig. 5.   Time course for the reaction of E·F21:HF31 with ATP in the presence of competitor DNA. 50 nM E·F21:HF31 was reacted with 2000 µM ATP in the presence of 4.0 µM competitor (dU)18. The formation of F21 and HF31 was monitored with the stopped-flow spectrofluorometer with lambda ex = 492 nm and lambda em >530 nm. Equations 5 and 6 were fitted to the data to give the following parameter values: a = 0.78 s, b = 0.26 s-1, c = 0 (fixed), d = -0.001, and e = 1 (fixed). The early phase of the reaction was attributed to unwinding the duplex DNA. The late phase of the reaction was attributed to complete strand separation with separation of the fluorescent probes.

Kinetics for Complete Strand Separation of F21:HF31 with [E] >>  [F21:HF31]-- To eliminate the contributions to the fluorescence signal due to dissociation of E·HF31, the reaction of F21:HF31 with E and ATP was repeated in the absence of competitor DNA and with the enzyme concentration (>300 nM) much greater than the Km of E·ATP for HF31 (30 nM). At the end of this reaction the strands of F21:HF31 were completely separated, and HF31 was bound to the enzyme. A typical time course for the fluorescence increase resulting from formation of F21 catalyzed by 348 nM helicase with 1000 µM ATP and 12.5 nM F21:HF31 is presented in the inset of Fig. 6. The fluorescence of the solution did not increase significantly until 1 s after mixing the components. This lag was not highly dependent on the order of addition of enzyme, ATP and F21:HF31. After the lag phase, the fluorescence increased in a first-order process. Equations 5 and 6 were fitted to these data to give a lag time of 1.14 ± 0.05 s and a first-order rate constant of 0.255 ± 0.002 s-1 (Fig. 6). The length of the lag phase was independent of enzyme concentration, whereas the value of the pseudo first-order rate constant for the late phase of the reaction (kobs) was highly dependent on enzyme concentration. The value of kobs for saturating helicase concentration with 2000 µM ATP was 0.50 ± 0.03 s-1. This was the maximal value for the effective rate constant for strand separation at infinite enzyme concentration (keff). The concentration of enzyme that gave 50% of this value was 360 ± 60 nM. With enzyme and F21:HF31 concentrations fixed at 200 and 12.5 nM, respectively, the dependence of kobs on ATP concentration was described by Equation 3 (k-1 = 0) with k1 = 0.217 ± 0.008 s-1 and K = 350 ± 40 µM.


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Fig. 6.   Reaction of E·F21:HF31 with HCV helicase and ATP in the absence of competitor (dU)18. The time course for the reaction of 348 nM helicase with 1000 µM ATP and 12.5 nM F21:HF31 is given in the inset. Equations 5 and 6 were fitted to the data to give the following values for the parameters: a = 1.14 s, b = 0.255 s-1, c = 0 (fixed), d = 0.2, and e = 1 (fixed). These values were used to calculate the solid line. The pseudo first-order rate constant for the late phase of the reaction with 2000 µM ATP was determined as a function of enzyme concentration. Equation 3 (k-1 = 0) was fitted to these data with k1 = 0.50 s-1and K = 360 nM.

Early Phase of the Reaction of E·ATP with 21:HF31 or 42:HF52 Was Unwinding of the Duplex-- Fluorescence changes were also observed (lambda ex = 492 nm and lambda em >530 nm) during the reaction of E·ATP with 21:HF31. Because this substrate lacked the fluorescein moiety on the 21-mer, these fluorescence changes were the result of changes in the environment of HF31 during the unwinding reaction. Experiments with free HF31 demonstrated that E·ATP quenched the fluorescence of HF31 (lambda ex = 510 nm and lambda em = 552 nm) by 55%. In contrast to the results observed with HF31, the fluorescence of HF31 in duplex 21:HF31 was not affected significantly by binding to E·ATP. Consequently, the unwinding of 25 nM 21:HF31 in the presence of excess enzyme ([E] > Km of E·ATP for HF31) was predicted to quench the fluorescence of HF31 in 21:HF31 only after separation of HF31 from 21 (Fig. 7). The enzyme initially formed E·21:HF31 with little fluorescence quenching. Subsequently, there was a small decrease in HF31 fluorescence that was linearly dependent on time. The late phase of the reaction was a first-order large decrease (~30%) in HF31 fluorescence. Equations 5 and 6 were fitted to the data collected in the presence of 1000 µM ATP to yield a = 1.78 ± 0.03 s, b = 0.427 ± 0.005 s-1, c = 1 (fixed), d = 0.865 ± 0.009, and e = 0 (fixed). The early phase of this reaction was attributed to duplex unwinding, whereas the late phase represented strand separation with concomitant isomerization of E·HF31 to a less fluorescent complex.


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Fig. 7.   Effect of the length of duplex DNA on the lag in product formation. Time course for the reaction of 475 nM helicase with 25 nM 21:HF31 (left tracing) or 25 nM 42:HF52 (right tracing) in the presence of 1000 µM ATP. Fractional fluorescence changes were normalized to the total fluorescence change observed for each reaction (33 and 30% fluorescence quenching for 21:HF31 and 42:HF52, respectively). Equations 5 and 6 were fitted to the data for the reaction of 21:HF31 with the enzyme to give the following values for the parameters: a = 1.78 s, b = 0.427 s-1, c = 1 (fixed), d = 0.865, and e = 0 (fixed). The values for the parameters for the reaction of 42:HF52 with the enzyme were a = 4.32 s, b = 0.119 s-1, c = 1 (fixed), d = 0.898, and e = 0 (fixed).

This interpretation was verified by demonstrating that the lag time (a) was related to the length of the duplex substrate. In particular, the lag time determined for unwinding 42:HF52 was approximately twice as long as that for 21:HF31 (Fig. 7). The 42-mer was a tandem duplication of the sequence of the 21-mer, and the complementary 52-mer was analogous to HF31. Equations 5 and 6 were fitted to the data for the reaction of 25 nM 42:HF52 with 475 nM helicase (Fig. 7) to yield a = 4.32 ± 0.05 s, b = 0.1195 ± 0.0006 s-1, c = 1 (fixed), d = 0.898 ± 0.005, and e = 0 (fixed). The lag phase was 2.4-fold longer with the 2-fold longer duplex, and the value of the pseudo first-order rate constant was 25% that for the shorter double-stranded DNA. Thus, the direct correlation between the length of the double-stranded DNA and the length of the early phase supported the interpretation that continuous unwinding of double-stranded DNA occurred in the early phase of the reaction.

Dissociation of F21 and HF31 from E-DNA Complexes-- The value of kcat for strand separation of F21:HF31 by HCV helicase was 0.12 s-1, which was significantly less that the maximal value for the effective rate constant for strand separation in a half-reaction of E·F21:HF31 with ATP (0.5 s-1, Fig. 6). Consequently, another kinetic step such as dissociation of product (F21 or HF31) from the enzyme-DNA complex was a major contributor to kcat. This possibility was also suggested from differences in the time course for formation of enzyme-free F21 and enzyme-free HF31 (Fig. 8A). In these experiments, E·F21:HF31 was reacted with 2000 µM ATP and 200 µg/ml poly(rU). F21 was monitored by the fluorescence increase with lambda ex = 492 nm and lambda em = 522 nm. HF31 was monitored by the fluorescence increase with lambda ex = 550 nm and lambda em = 590 nm. The latter wavelengths minimized but did not eliminate the contribution of F21 fluorescence to the HF31 fluorescence signal. The fluorescence changes were normalized to the total fluorescence change for formation of enzyme-free F21 and enzyme-free HF31. The time courses of the reaction with these monitoring conditions were different (Fig. 8A). The increase in fluorescence from F21 (lambda ex = 492 nm) was a single exponential process (k1 = 0.57 ± 0.01 s-1 and F1 = -0.2227 ± 0.0006). The increase in fluorescence from F21 and HF31 (lambda ex = 550 nm) was a double-exponential process (k1 = 0.57 s-1 (fixed), k2 = 0.18 ± 0.03 s-1, F1 = 0.10 ± 0.07, and F2 = -0.43 ± 0.06). The early phase of this time course was attributed to the fluorescence change associated with F21, whereas the late phase was attributed to fluorescence changes associated with HF31. These results suggested that dissociation of E·HF31 was the last step of the catalytic cycle (Equation 10) (8).


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Fig. 8.   Dissociation of E·HF31 in the presence of ATP. A, comparison of the time courses for formation of free F21 and free HF31 from E·F21:HF31. ATP (2000 µM) with 200 µg/ml poly(rU) was added to 50 nM E·F21:HF31. F21 was monitored with lambda ex = 492 nm and lambda em = 550 nm, and HF31 was monitored with lambda ex = 550 nm and lambda em = 590 nm. The solid line for the lower tracing was drawn with a first-order rate constant of 0.57 s-1; the solid line for the upper tracing was drawn with first-order rate constants of 0.57 s-1 and 0.18 s-1. B, effect of ATP on the dissociation of E·HF31. The dissociation of E·HF31 was monitored with the stopped-flow spectrofluorometer by the fluorescence increase upon formation of HF31 (lambda ex = 492 nm and lambda em >530 nm). If the trap (5 µM 45-mer) and 700 µM ATP were added simultaneously to E·HF31 (generated by reaction of 50 nM E and 150 nM HF31 in the presence or absence of ATP), the time course of the reaction was distinctly biphasic (inset, 0.64 s-1 and 0.19 s-1). The dependence of the smaller rate constant on ATP concentration was determined from data collected by simultaneous addition of ATP and trap to E·HF31. Equation 3 was fitted to these data with k-1 = 0.009 s-1, K = 130 µM, and k1 = 0.21 s-1 (solid line).

To confirm that dissociation of E·HF31 was the late phase in this reaction, the values of the rate constants for dissociation of F21 and HF31 from the enzyme-DNA complex were determined. The dissociation of E·HF31 and E·F21 was monitored by the fluorescence changes (lambda ex = 492 nm and lambda em >530 nm) associated with formation of free HF31 and F21. Free enzyme was trapped with excess (5 µM) 31-mer or 45-mer (TTT TTT ACA ACG TCG TGA CTC TCT CTC TCT CTC TCT CTC TCT CTC). Both of these trapping agents yielded similar results at several concentrations. In the absence of ATP, the fluorescence change was a first-order process. The values of the dissociation rate constants for E·F21 and E·HF31 were 0.0345 ± 0.0004 s-1 and 0.009 ± 0.001 s-1, respectively, which were too small for these processes to be competent in steady-state catalysis. If the competitor DNA was added simultaneously with 2000 µM ATP to E·F21 or E· HF31, the fluorescence change was biphasic (Fig. 8B, inset). In the case of E·HF31, addition of ATP to E·HF31 to form "E·HF31·ATP" prior to initiating the reaction with competitor DNA essentially eliminated the initial phase of the reaction. In the absence of a DNA competitor to trap E as E·HF31 dissociated, the amplitude of the late phase of the reaction was very small (data not shown). Based on these observations, the early phase of the fluorescence change was assigned to the reaction of E·HF31 with ATP to form E·HF31·ATP as described by Equation 11. The late phase of the fluorescence change was assigned to the subsequent dissociation of "E·HF31·ATP" to form E·ATP·DNA, where DNA was the competitor DNA. The kinetics of the early phase of the reaction of ATP with E·HF31 were described by Equation 3 (k-1 = 0) with K = 150 ± 40 µM and k1 = 1.6 ± 0.2 s-1.

The first-order rate constants for dissociation of E·HF31·ATP and E·F21·ATP (late phase of the reaction) at a saturating concentration of ATP were 0.202 ± 0.003 s-1 and 1.52 ± 0.01 s-1, respectively. The value of the rate constant for dissociation of E·HF31·ATP was similar to the value of kcat for unwinding F21:HF31 (0.12 s-1, Fig. 2), whereas the value of the rate constant for dissociation of E·ATP·F21 was much too large to contribute significantly to kcat.

Because the release of HF31 from E·HF31 could be a major contributor to kcat for the unwinding of F21:HF31, the effect of ATP concentration on the rate constant for dissociation of E·HF31 (late phase of the reaction, inset Fig. 8B) was investigated in more detail (Fig. 8B). These data were described by Equation 3 with k-1,ATP = 0.21 ± 0.01 s-1, K = 90 ± 20 µM, and k-1 = 0.009 s-1 (fixed), where k-1,ATP (k1 in Equation 3) was the dissociation constant of E·HF31·ATP and k-1 was the dissociation constant of E·HF31.

F21:HF31 and 21:31 Interact with HCV Helicase Similarly-- F21:HF31 was the double-stranded DNA used for most of the experiments described herein. To ensure that our results were not significantly affected by the fluorescence labels on F21:HF31, selected kinetic parameters were determined for the unlabeled duplex 21:31 (Table II). Comparison of the kinetic parameters for the reaction of enzyme with F21:HF31 and 21:31 (Table II) demonstrated that labeled and unlabeled double-stranded DNA were interacting similarly with the enzyme.

                              
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Table II
Comparison of selected kinetic parameters for the reaction of HCV helicase with F21:HF31, 21:31, and HF31
Kinetic parameters are defined in Equation #9.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Crystal structures for helicases from Bacillus stearothermophilus (36), HCV (37), and E. coli (38) in the absence of divalent metal ion indicated that they were monomeric in the crystalline state. However, many helicases, such as E. coli Rep helicase (4), T4 helicase (32), and DnaB helicase (39), appear to be oligomeric in the presence of substrates. In contrast to these helicases, HCV helicase was a monomeric protein in the presence of DNA and/or ATP analogues. Because HCV helicase contains a single DNA-binding site per monomer (22), these results indicated that the active form of this enzyme had a single DNA-binding site. Helicases have been proposed to unwind duplex nucleic acids by either an "active" or "passive" mechanism (7). The former mechanism requires that the catalytically active form of the enzyme has multiple nucleic acid-binding sites. Our finding that the active form of HCV helicase was monomeric with a single nucleic acid-binding site suggested that HCV catalyzed the unwinding of F21:HF31 by a passive mechanism.

The goal of the present study was to identify the kinetic steps that contributed to kcat. Unfortunately, relating pre-steady-state data to the steady-state kcat was complicated by the processivity of the enzyme, by the step size, and by the number of base pairs broken for each ATP consumed. For helicases, a simplified scheme describing the unwinding of duplex DNA ((bp)n) bound to the enzyme is given by Equation 12.
<AR><R><C>E·(<UP>bp</UP>)<SUB>n</SUB></C><C><LIM><OP><ARROW>→</ARROW></OP><UL>k</UL></LIM></C><C>E·(<UP>bp</UP>)<SUB>n<UP>−</UP>(n/m)</SUB></C><C><LIM><OP><ARROW>→</ARROW></OP><UL>k</UL></LIM></C><C>E·(<UP>bp</UP>)<SUB>n<UP>−</UP>(2n/m)</SUB></C><C><LIM><OP><ARROW>→</ARROW></OP><UL>k</UL></LIM> <LIM><OP><ARROW>→</ARROW></OP><UL>k</UL></LIM></C><C>E·(<UP>bp</UP>)<SUB>0</SUB></C></R><R><C>↓ k′</C><C></C><C>↓ k′</C><C></C><C>↓ k′</C><C></C><C>↓ k′</C></R></AR> (Eq. 12)
In this simplified mechanism, the unwinding of double-stranded DNA with n base pairs (bp) was considered to occur sequentially in n/m steps with each unwinding event described by the pseudo first-order rate constant (k) that could be dependent on ATP concentration. Furthermore, each partially unwound duplex could dissociate from the enzyme and anneal in a process described by the rate constant k'. This model is analogous to that described by Ali and Lohman for E. coli helicase II (14). The processivity of the enzyme for DNA (P) in this model is given by Equation 13. The fraction of double-stranded DNA that was separated into single-stranded DNA by HCV helicase in a single binding event is given by Equation 14.
P=<FR><NU>k</NU><DE>k+k′</DE></FR> (Eq. 13)
<UP>fractional DNA unwound</UP>=<FENCE><FR><NU>k</NU><DE>k+k′</DE></FR></FENCE><SUP><FR><NU>n</NU><DE>m</DE></FR></SUP> (Eq. 14)
An explicit expression for the time courses of strand separation has been derived for the simple model of Equation 12 (14). In this model, the reaction was initiated by addition of ATP and competitor DNA to E·(bp)n. Competitor DNA ensured that partially unwound (bp)n that dissociated from the enzyme annealed and did not rebind to the enzyme. The time course for strand separation of (bp)n (f(t)) is given by Equation 15.
f(t)=P<SUP><FR><NU>n</NU><DE>m</DE></FR></SUP><FENCE>1−<FR><NU><LIM><OP>∑</OP><LL>s<UP>=</UP>0</LL><UL><FR><NU>n</NU><DE>m</DE></FR></UL></LIM>((k+k′)t)<SUP>s</SUP>e<SUP><UP>−</UP>(k<UP>+</UP>k′)t</SUP></NU><DE>s!</DE></FR></FENCE> (Eq. 15)
The assumptions for this derivation corresponded to experimental conditions for the data of Fig. 5 where a competitor DNA was present to trap free enzyme as F21:HF31 or products dissociated from the enzyme. Numerical simulation of the time courses for strand separation of a 24-mer were made with step sizes equal to 1, 2, 3, 4, 6, 8, 12, and 24. A 24-mer was chosen over a 21-mer for these simulations because of the greater number of possible step sizes evenly divisible into 24. The simulated time courses for strand separation were biphasic in all cases except for a step size of 24 base pairs. The initial phase of the reaction using small step sizes was approximately linearly dependent on time, and the late phase of the reaction was exponentially dependent on time. The lag time for the reaction (the time defined by the intersection of the line through the lag phase data and the line drawn with the maximal slope through the exponential phase data (tl in Fig. 5)) was directly proportional to the number of steps. More importantly, the time for transition from the linear phase to the exponential phase (the portion of tl that the time course had definite curvature, tt in Fig. 5) was a decreasing fraction of the lag time of the reaction. For example, the transition from the linear phase to the exponential phase for the simulated data was 26, 46, and 62% of the lag time for step sizes of 1, 2, and 3 base pairs, respectively. The transition from the linear phase to the exponential phase for unwinding F21:HF31 and 21:HF31 (Fig. 5 and Fig. 7) was very abrupt (~25% of the total lag time). Based on the simple model above, these results suggested that HCV helicase disrupted F21:HF31 at most several base pairs at a time. Because the free energy of hydrolysis of ATP is sufficient to break 2 to 4 base pairs (2, 7, 25), it was not unreasonable to propose a step size of 2. This would result in the efficiency for coupling the energy of ATP hydrolysis to base pair breaking of over 50%. For a step size of 2 base pairs, a lag time of approximately 1 s (average value from the data of Fig. 5 and 6), and a duplex of 21 base pairs (F21:HF31), the value for the sum of k and k' in Equation 12 was estimated from the simulations described above to be approximately 7.5 s-1. From the value for the sum of k and k' (7.5 s-1), the step size (m = 2), the fraction unwound (0.44), and Equation 14, the values of k and k' were estimated to be 7.0 s-1 and 0.5 s-1, respectively. The calculated value for dissociation of F21:HF31 in various stages of unwinding from E·ATP·F21:HF31 (k' = 0.5 s-1) was in good agreement with the experimental measured value of 0.84 s-1 (Fig. 3). Furthermore, the values for P, n/m, and k were used to predict the maximal value for the effective rate constant (keff) for formation of F21 from E·ATP·F21:HF31 in the presence of a saturating concentration of enzyme (Fig. 6). The expression for keff for conversion of (bp)n to (bp)0 derived for the model of Equation 12 with steady-state assumptions is given by Equation 16.
k<SUB><UP>eff</UP></SUB>≈<FR><NU>(P−1)P<SUP><FENCE><FR><NU>n</NU><DE>m</DE></FR><UP>−</UP>1</FENCE></SUP>k</NU><DE>P<SUP><FR><NU>n</NU><DE>m</DE></FR></SUP>−1</DE></FR> (Eq. 16)
Substituting estimated values for n/m (~11), k (7 s-1), and P (0.93) into this expression, the predicted value for keff was 0.43 s-1, which was similar to the observed value of 0.50 s-1 (Fig. 6).

Because the maximal value of the pseudo first-order rate constant for formation of F21 from F21:HF31 with excess E and ATP (0.5 s-1) was 4-fold larger than the value of kcat (0.12 s-1), another step must contribute significantly to kcat. The dissociation rate constant of E·ATP·HF31 was 0.21 s-1, which was similar to kcat. If product dissociation and the unwinding step were the sole contributors to kcat, the calculated value of kcat (0.15 s-1) was very close to the experimental value (0.12 s-1). Thus, the dissociation of E·HF31 was the major contributor to kcat for strand separation of F21:HF31 and the unwinding steps were minor contributors to kcat. The relevance of the interpretation of the kinetic results presented herein for F21:HF31 to normal substrate was dependent on the assumption that the tagged and untagged DNA substrates interacted similarly with the enzyme. The similarity of the selected kinetic parameters for interaction of the tagged and untagged DNA molecules with the enzyme (Table II) suggested that this assumption was valid.


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    ACKNOWLEDGEMENT

We gratefully acknowledge E. Furfine for helpful discussions during the course of these studies.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Glaxo Wellcome, 5 Moore Dr., Research Triangle Park, NC 27709. Tel.: 919-483-4390; Fax: 919-483-3895.

1 The abbreviations used are: HCV, hepatitis C virus; HCV helicase, truncated domain of the NS3 protein including amino acid residues 1193-1657 of the HCV genotype 1b polyprotein; MOPS, 3-N-morpholinopropanesulfonic acid; E, helicase; E·ATP, binary complex formed from E and ATP that may be mixtures of ATP, ADP, and inorganic phosphate; E·DNA, complex between E and DNA; and E·DNA·ATP, ternary complex of helicase, DNA, and ATP that may be mixtures of ATP, ADP, and inorganic phosphate with an undefined fraction of the base pairs in the duplex DNA disrupted; F, chemically modified fluorescein as defined in Oligos Etc. catalogue; HF, chemically modified hexachlorofluorescein as defined in Oligos Etc. catalogue; 21-mer, GAG TCA CGA CGT TGT AAA AAA; 31-mer, TTT TTT ACA ACG TCG TGA CTC TCT CTC TCT C; F21, GAG TCA CGA CGT TGT AAA AAA-F; HF31, HF-TTT TTT ACA ACG TCG TGA CTC TCT CTC TCT C; 42-mer, GAG TCA CGA CGT TGT AAA AAA GAG TCA CGA CGT TGT AAA AAA; HF52, HF-TTT TTT ACA ACG TCG TGA CTC TTT TTT ACA ACG TCG TGA CTC TCT CTC TCT C; I, stem-loop structure, GGC CTA AGC GTA TCG CTT AGG CCG AGT CAG G; I-F, I with fluorescein on the 3' end; AMPCPP, alpha ,beta -methyleneadenosine 5'-triphosphate; ATPgamma S, adenosine 5'-O-(3-thiotriphosphate); ADPPNP, 5'-adenylimidodiphosphate; bp, base pair.

2 The value of the dissociation constant for E·I estimated from sedimentation data (<1 µM) was significantly higher than that calculated from fluorescence titration experiments (1.3 ± 0.4 nM). This may be due to differences in experimental conditions. Sedimentation equilibrium data were collected at 4 °C, whereas the fluorescence titration data and the steady-state kinetic data were at 25 °C. Helicase and I concentrations were also 50-500-fold greater in the sedimentation experiments. Sedimentation equilibrium data are a direct measure of molecular mass, and dissociation constants derived from these data are directly influenced by the molecular mass distribution. There was a possibility that a small fraction of helicase was not competent to bind I and/or that a small fraction of I was not competent to bind the enzyme. In each case, the amount of monomeric helicase measured in the sedimentation experiment would be artificially large. This additional "free" helicase would sediment as monomeric protein and would result in a higher apparent dissociation constant. If an allowance were made to accommodate 15-20% excess non-interacting monomer, the resulting dissociation constant would be similar to that measured by fluorescence titration.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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