Inhibition of the Hepatitis C Virus Helicase-associated ATPase Activity by the Combination of ADP, NaF, MgCl2, and Poly(rU)
TWO ADP BINDING SITES ON THE ENZYME-NUCLEIC ACID COMPLEX*

David J. T. PorterDagger

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 has an intrinsic ATPase activity and a nucleic acid (poly(rU))-stimulated ATPase activity. The poly(rU)-stimulated ATPase activity was inhibited by F- in a time-dependent manner during ATP hydrolysis. Inhibition was the result of trapping an enzyme-bound ADP-poly(rU) ternary complex generated during the catalytic cycle and was not the result of generating enzyme-free ADP that subsequently inhibited the enzyme. However, catalysis was not required for efficient inhibition by F-. The stimulated and the intrinsic ATPase activities were also inhibited by treatment of the enzyme with F-, ADP, and poly(rU). The inhibited enzyme slowly recovered (t1/2 = 23 min) ATPase activity after a 2000-fold dilution into assay buffer. The onset of inhibition by 500 µM ADP and 15 mM F- in the absence of nucleic acid was very slow (t1/2 > 40 min). However, the sequence of addition of poly(rU) to a diluted solution of ADP/NaF-treated enzyme had a profound effect on the extent of inhibition. If the ADP/NaF-treated enzyme was diluted into an assay that lacked poly(rU) and the assay was subsequently initiated with poly(rU), the treated enzyme was not inhibited. Alternatively, if the treated enzyme was diluted into an assay containing poly(rU), the enzyme was inhibited. ATP protected the enzyme from inhibition by ADP/NaF. The stoichiometry between ADP and enzyme monomer in the inhibited enzyme complex was 2, as determined from titration of the ATPase activity ([ADP]/[E] = 2.2) and from the number of radiolabeled ADP bound to the inhibited enzyme ([ADP]/[E] = 1.7) in the presence of excess NaF, MgCl2, and poly(rU). The Hill coefficient for titration of ATPase activity with F- (n = 2.8) or MgCl2 (n = 2.1) in the presence of excess ADP and poly(rU) suggested that multiple F- and Mg2+ were involved in forming the inhibited enzyme complex. The stoichiometry between (dU)18, a defined oligomeric nucleic acid substituting for poly(rU), and enzyme monomer in the inhibited enzyme complex was estimated to be 1 ([(dU)18/[E] = 1.2) from titration of the ATPase activity in the presence of excess ADP, MgCl2, and NaF.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Hepatitis C virus (HCV)1 genome encodes for an RNA helicase that presumably is essential for viral replication (1). Helicases catalyze the separation of double-stranded nucleic acids into single-stranded nucleic acids with the concomitant hydrolysis of ATP (2). The mechanism of coupling of ATP hydrolysis to double-stranded nucleic acid unwinding is unclear (2, 3). Escherichia coli Rep helicase is the most thoroughly characterized helicase in terms of characterizing the binding of DNA and the associated ATPase activities (3-8). Recently, a crystal structure of the Rep helicase complexed with DNA and ADP has been reported. Helicase motifs Ia, V, and III were implicated in single-stranded DNA binding, motifs I and IV were involved in nucleotide binding, and motifs II and IV possibly functioned in the coupling of nucleotide and single-stranded DNA binding (9). Unfortunately, the protein was crystallized in the absence of a divalent metal cofactor such as Mg2+, which is required for ATP hydrolysis and nucleic acid unwinding. Crystal structures of DNA helicase from Bacillus stearothermophilus (10) and of RNA helicase from HCV have also been reported. In neither case was the divalent metal cofactor present. Nonetheless, these structures suggested a single nucleotide binding site per monomer.

HCV helicase has an intrinsic ATPase activity and an nucleic acid-stimulated ATPase activity. Single-stranded DNA or RNA increased the kcat value of HCV helicase ATPase activity, at most, 50-fold (12). For example, poly(rU) enhanced kcat 30-fold. In contrast, the kcat values for ATPase activities of bacterial helicases such as Rep helicase and helicase II are stimulated over a 1000-fold by single-stranded DNA (8). Turnover numbers for HCV helicase and Rep helicase determined in the presence of single-stranded nucleic acid are similar (8, 12). The differential DNA stimulation of these enzymes is the result of the large intrinsic ATPase activity of HCV helicase in the absence of single-stranded nucleic acid. This observation suggests that either HCV helicase ATPase activity is associated with a single site that is not tightly coupled to nucleic acid binding or HCV helicase may have ATPase activity associated with two separate sites on the enzyme. The latter possibility could be verified by determining the stoichiometry for ATP binding to the enzyme. However, the ATPase activity of the enzyme would require nonhydrolyzable ATP analogues for titration of ATP sites. Unfortunately, the affinity of the enzyme for nonhydrolyzable ATP analogues was very low, e.g. the Ki for ADPNP was 200 µM (12), which was too high for stoichiometry determination.

The complex between F- and Al3+ or Be2+ is an alternative class of inhibitors that is useful for mechanism studies with phosphotransferases. These complexes, which mimic phosphate, bind tightly to numerous nucleoside triphosphatases in the presence of the nucleotide diphosphate (13-16). However, some phosphotransferases effectively bind nucleotide diphosphate in the presence of F- and Mg2+ without Al3+ or Be2+ as a cofactor (17-23). In the present studies, the ATPase activity of HCV helicase was inhibited with the combination of ADP, F-, MgCl2, and poly(rU) in the absence of Al3+ or Be2+. The stoichiometry between ADP and HCV helicase monomer in the inhibited complex was 2, which suggested two distinct nucleotide triphosphate binding sites on the enzyme.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Rabbit muscle pyruvate kinase, rabbit muscle lactate dehydrogenase, NADH, ATP, phosphoenolpyruvate, deferoxamine mesylate, ADP, CDP, UDP, GDP, 2'-deoxyADP, adenosine-2,8-3H 5'-diphosphate (25.2 Ci/mmol, >93% purity), and MOPS were from Sigma. The concentration of ADP in solution was estimated with epsilon 258 = 14.4 mM -1 cm-1. Poly(rU) and poly(rA) were Amersham Pharmacia Biotech products. The concentration of nucleobase in solutions of poly(rU) and poly(rA) was estimated with an epsilon 260 = 9.35 mM-1 cm-1 and epsilon 258 = 9.80 mM-1 cm-1 (per nucleobase), respectively. Gel-purified 16-mer (TTT TTT ACA ACG TCG T) and 45-mer (GTT TTT TAC AAC GTC GTG ACT CTC TCT CTC TCT CTC TCT CTC TCT) were from Oligos Etc. The concentration of these oligomers were estimated with epsilon 260 = 147 mM-1 cm-1 and 379 mM-1 cm-1, respectively. NaF and AlF3 were of the highest purity from Aldrich. HCV helicase was purified as described previously (12). The concentration of HCV helicase monomer was calculated with an epsilon 280 = 88.5 mM-1 cm-1 (12), which is based on (rU)15 binding sites. The standard buffer was 0.05 M MOPS, 3.5 mM MgCl2 at pH 7.0.

Assay of ATPase Activity-- ATPase activity was assayed at 25 °C by monitoring the formation of ADP spectrophotometically at 340 nm. The absorbance change was due to the oxidation of NADH that was coupled to the phosphorylation of ADP through pyruvate kinase and lactate dehydrogenase as described previously (12). The standard assay was 10 units/ml pyruvate kinase, 10 units/ml lactate dehydrogenase, 350 µM NADH, 2.0 mM phosphoenolpyruvate, 1.0 mM ATP, and 100 µg/ml poly(rU) prepared in the standard buffer. The definition of a unit of pyruvate kinase or lactate dehydrogenase was that of the supplier. The spectrophotometric ATPase assay was initiated by two methods. For the first method (Method Ia), the assay was initiated by dilution (1:100) of the enzyme directly into the standard assay containing poly(rU). For the second method (Method Ib), treated enzyme was diluted (1:100) into the standard assay lacking poly(rU). This solution was equilibrated for 60 s to allow dissociation of readily reversible complexes of enzyme with inhibitor. The assay was initiated with 100 µg/ml poly(rU). The "fluorokinase" activity associated with pyruvate kinase (24) was not significant under these assay conditions.

Adenosine-2,8-3H 5'-Diphosphate Binding to Helicase-- The enzyme was incubated at 21 °C in the standard buffer with 30 mM NaF, 200 µg/ml poly(rU), and [2,8-3H]ADP (46 µCi/µmol) for 20 min. The enzyme-bound nucleotide was separated from free nucleotide by size exclusion chromatography on a centrifuge column (1 ml disposable syringe filled with Bio-Rad P-6 resin equilibrated in the standard buffer at 21 °C analogous to the method of Penefsky (25)). The bound nucleotide was quantitated by liquid scintillation counting using ICN EcoLume scintillation mixture.

Data Analysis-- The time course for hydrolysis of ATP to ADP (P(t)) was described by Equation 1, where Vi was the initial velocity of the reaction, Vf was the final velocity of the reaction, and kobs was the apparent first-order rate constant for transition from Vi to V.
P(t)=<FR><NU>V<SUB><UP>i</UP></SUB>−V<SUB>f</SUB></NU><DE>k</DE></FR>(1−e<SUP><UP>−</UP>k<SUB><UP>obs</UP></SUB>t</SUP>)+V<SUB>f </SUB>t (Eq. 1)

The dependence on NaF concentration of the uncorrected kobs values (uncorrected for inhibition of initial velocity by NaF) for inhibition was described by a linear function (Equation 2). The initial velocity of ATP hydrolysis was inhibited significantly by NaF. It was assumed that the rate constant for onset of inhibition was directly proportional to the rate of ATP hydrolysis. Consequently, kobs values were dividing by the fractional initial velocity in the presence of NaF to yield corrected kobs values. The dependence of the corrected kobs values on NaF concentration was described by an exponential function (Equation 3). The value of n was an indication of the number of NaF molecules involved in the inhibition process.
k<SUB><UP>obs</UP></SUB>=ax (Eq. 2)
k<SUB><UP>obs</UP></SUB>=ax<SUP>n</SUP> (Eq. 3)

The effect of pyruvate kinase concentration on kobs was described by a linear function (Equation 4), where the concentration of pyruvate kinase was expressed in units of enzyme/ml. A unit of enzyme was based on the value supplied by the manufacturer.
k<SUB><UP>obs</UP></SUB>=a[<UP>pyruvate kinase</UP>]+b (Eq. 4)

Titration of helicase (E) with ADP in the presence of excess F- and Mg2+ was monitored by the decrease in ATPase activity. It was assumed that the titration could be described by the simplified mechanism shown in Reaction 1, where K was the dissociation constant of E for ADP.
E+<UP>ADP</UP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>K</UL></LIM> E · <UP>ADP</UP>
<UP><SC>Reaction</SC> 1</UP>
It was also assumed that the fractional activity remaining after treatment (A([ADP])) was related to the total concentration (sum of free and bound) of added ADP ([ADP]) by Equation 5, in which Delta Ainfinity was the fractional activity decrease resulting from conversion of E to E·ADP.
A([<UP>ADP</UP>])=1−&Dgr;A<SUB>∞</SUB><FR><NU>[E · <UP>ADP</UP>]</NU><DE>[E<SUB><UP>t</UP></SUB>]</DE></FR> (Eq. 5)

Et was the total concentration (sum of free and bound) of ADP binding sites on the enzyme (i.e. for n sites per monomer Et was n[E], where [E] was the molar concentration of enzyme monomer). Because the concentration of enzyme was comparable to the dissociation constant of the enzyme for ADP, the concentration of E·ADP was related to [ADP] by Equation 6.
[E · <UP>ADP</UP>]=<FR><NU>[<UP>ADP</UP>]+[nE]+K</NU><DE>2</DE></FR>−<FR><NU>1</NU><DE>2</DE></FR> <RAD><RCD>([<UP>ADP</UP>]+[nE]+K)<SUP>2</SUP>−4[nE][<UP>ADP</UP>]</RCD></RAD> (Eq. 6)

If more than one binding site for ADP was present on the enzyme, the concentration of E was multiplied by n, where n was a fitted parameter. This assumed that the ADP bound to each site on E independently. If the fit of Equations 5 and 6 to the data was significantly better when n was greater than 1, the calculated "dissociation constant (K)" of E for ADP was the concentration of free ADP, for which 50% of E was in the E·ADP form. The value of n from this fitting routine was an estimate of the number of binding sites on the enzyme for ADP.

The time courses for inhibition of helicase in the absence of poly(rU) were described by Equation 7, where A(t) was the normalized activity (initial velocity) at time equal to t, Af was the activity at the end of the reaction, and k was the pseudo first-order rate constant for transition from the initial activity (1.0) to the final activity (Af).
A(t)=A<SUB>f</SUB>+(1−A<SUB>f</SUB>)e<SUP><UP>−</UP>kt</SUP> (Eq. 7)

Titration of ATPase activity with MgCl2 or NaF in the presence of fixed concentrations of poly(rU) and ADP was described by the logistic equation (Equation 8), where A([L]) was the ATPase activity at a ligand concentration of [L] (L was NaF or MgCl2), and Delta Ainfinity was the maximal decrease in activity at an infinite concentration of L.
A([L])=1−<FR><NU>&Dgr;A<SUB>∞</SUB>[L]<SUP>n</SUP></NU><DE>K<SUP>n</SUP>+[L]<SUP>n</SUP></DE></FR> (Eq. 8)

Titration data for inhibition of ATPase activity by (dU)18 in the presence of excess NaF, ADP, and Mg2+ was modeled by Equations 9 and 10 for irreversible binding of an inhibitor to an enzyme. The ATPase activity at a total concentration of (dU)18 was normalized to that of untreated enzyme to give A([(dU)18]).
A([(<UP>dU</UP>)<SUB>18</SUB>])=1−<FENCE><FR><NU>1−b</NU><DE>a</DE></FR></FENCE>[(<UP>dU</UP>)<SUB>18</SUB>], [(<UP>dU</UP>)<SUB>18</SUB>]<a (Eq. 9)
A([(<UP>dU</UP>)<SUB>18</SUB>])=b, [(<UP>dU</UP>)<SUB>18</SUB>]≥a (Eq. 10)
b was the final ATPase activity, and a was concentration of (dU)18 that stoichiometrically inhibited the ATPase activity.

General Methods-- Absorbance and conventional kinetic data were obtained from a Uvikon 860 spectrophotometer at 25 °C or on a Spectramax 250 plate reader at 21 °C. 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

Inhibition of the ATPase Activity of Helicase by the Combination of ATP, NaF, and Poly(rU)-- The ATPase activity of helicase (>200 nM) was stable at room temperature in the standard buffer for over 30 min. However, the enzyme (230 nM) lost >95% of its ATPase activity after a 10-min reaction with 25 mM NaF, 1.25 mM ATP, and 250 µg/ml poly(rU). If the enzyme was reacted with NaF and ATP in the absence of poly(rU), the ATPase activity was not affected significantly (<10% inhibition). The inhibited enzyme slowly recovered activity (t1/2 ~ 20 min) after a 100-fold dilution into the standard assay. Thus, the time course for the onset of inhibition of helicase by 10 mM NaF, 2.0 mM ATP in the presence of 100 µg/ml poly(rU) could be readily monitored on a conventional spectrophotometer (Fig. 1). In the absence of NaF, the rate of hydrolysis of ATP was relatively constant, whereas in the presence of 10 mM NaF, the rate of ATP hydrolysis decreased exponentially (Fig. 1A). Equation 1 was fitted to the latter time course with Vi = 0.339 ± 0.003 µM s-1, kobs = (5.1 ± 0.2) × 10-3 s-1, and Vf = 0.018 ± 0.003 µM s-1. Each mol of enzyme hydrolyzed approximately 25,000 mol of ATP molecules during the inhibition process under these conditions. In contrast to these results, the intrinsic ATPase activity measured in the absence of poly(rU) was not significantly affected by 40 mM NaF (Fig. 1B).


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Fig. 1.   Time courses for inhibition of the helicase-associated ATPase activity by NaF and ATP. A, inhibition of poly(rU)-stimulated ATPase activity. The time course for hydrolysis of ATP by helicase (2.32 nM) in the standard assay solution with 2.0 mM and 100 µg/ml poly(rU) was linear (open symbols). In the presence of 10 mM NaF, the time course for hydrolysis of ATP was nonlinear (closed symbols). The solid line for the latter data was calculated by Equation 1 with Vi = 0.339 µM s-1, kobs = 5.1 × 10-3 s-1, and Vf = 0.018 µM s-1. B, inhibition of intrinsic ATPase activity. The time course for hydrolysis of ATP (2.0 mM) by helicase (92 nM) in the presence (closed symbols) or absence (open symbols) of 40 mM NaF was similar.

Selectivity of the Inhibition Reaction for Nucleic Acid Sequence-- Time courses similar to those of Fig. 1A for inhibition of the ATPase activity by 1.0 mM ATP and 30 mM NaF were determined with 300 µM (nucleobase) poly(rU), (dU)18, poly(A), a 45-mer, and a 16-mer as the trapping nucleic acid. Each of these nucleic acids caused a time-dependent inhibition of the ATPase activity in the presence of ATP. The pseudo first-order rate constants for onset of inhibition (kobs) were (8.3 ± 0.4) × 10-2 s-1, (3.6 ± 0.1) × 10-2 s-1, (6.9 ± 0.3) × 10-2 s-1, (3.51 ± 0.06) × 10-3 s-1, and (4.73 ± 0.06) × 10-4 s-1, respectively. Poly(rU) was selected for a more detailed investigation into the mechanism of inhibition of the ATPase activity.

Deferoxamine and Al3+ Did Not Affect the Pseudo First-order Rate Constant for Inhibition of ATPase Activity-- Inhibition of some ATPases by NaF has been attributed to formation of an aluminum or beryllium fluoride complex with enzyme-bound nucleotide diphosphate (15). Deferoxamine, a chelator of trivalent metal ions such as aluminum, diminishes the inhibitory effect of NaF and ADP on this class of enzymes, whereas AlF3 stimulates the inhibitory effect of NaF (15). However, deferoxamine or AlF3 had little effect on kobs for onset of inhibition of the HCV helicase ATPase activity in the standard buffer with 20 mM NaF, 100 µg/ml poly(rU), and 2.0 mM ATP. The value of kobs was 0.0153 ± 0.0002 s-1 in the absence of deferoxamine and 0.0136 ± 0.0003 s-1 in the presence of 1.0 mM deferoxamine. Similarly, the value of kobs was 0.0120 ± 0.0003 s-1 in the absence of AlF3, and 0.0157 ± 0.0004 s-1 in the presence of 400 µM AlF3. In the absence of poly(rU), 250 µM AlF3 and 10 mM NaF did not inhibit the ATPase activity significantly.

Selectivity of the Inhibition Reaction for the Metal Cofactor-- A divalent metal was required for the helicase-associated ATPase activity (25). Previous inhibition experiments used MgCl2 as the metal cofactor. Because MnCl2 can substitute for MgCl2 in the ATPase reaction with comparable efficiency (12), the inhibition experiments were repeated with 3.5 mM MnCl2 to determine the selectivity of the inhibitory process for the divalent metal cofactor. The kobs for inhibition of ATP hydrolysis by 2.4 nM helicase with 10 mM NaF in the presence of 2.0 mM ATP, 100 µg/ml poly(rU) and 3.5 mM MgCl2 was (6.5 ± 0.1) × 10-3 s-1. When MnCl2 was substituted for MgCl2, the value of kobs was (7.8 ± 0.2) × 10-4 s-1. The ratio of the kobs value for onset of inhibition with Mn2+ as the divalent metal cofactor to that with Mg2+ was 0.12.

Effect of NaF Concentration on kobs for Onset of Inhibition-- The value of the first-order rate constant for onset of inhibition of ATPase activity (kobs) was determined at varying NaF concentration with 2.0 mM ATP, 100 µg/ml poly(rU), 3.5 mM MgCl2, and 2-20 nM helicase. The value of kobs increased linearly with NaF concentration (Fig. 2, open symbols). Equation 2 was fitted to these data with a = 0.00113 ± 0.00006 µM-1 s-1. However, when the values of kobs were corrected for the decrease in initial velocity of ATP hydrolysis at high concentrations of NaF, the value of kobs increased exponentially with the concentration of NaF (Fig. 2, closed symbols). Equation 3 was fitted to these data with n = 1.77 ± 0.05 and a = (8 ± 2) × 10-5 µM-2 s-1. This value of n suggested that multiple NaF molecules were in the inhibited enzyme complex.


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Fig. 2.   Effect of NaF concentration on the onset of inhibition of ATPase activity. The time courses for hydrolysis of ATP by helicase (35-120 nM) were determined with 250 µg/ml poly(rU), 2.0 mM ATP, and the indicated concentrations of NaF in the standard assay buffer. Equation 1 was fitted to these data to obtain the pseudo first-order rate constant for onset of inhibition (kobs) and the initial velocity for ATP hydrolysis. The kobs values were approximately linearly dependent on NaF concentration (open symbols). The solid line was calculated with Equation 2 and a = 0.00113 µM-1 s-1. Corrected kobs values were exponentially dependent on NaF concentration. The solid line was calculated with Equation 3 and a = 8 × 10-5 µM-2 s-1and n = 1.77.

Effect on Pyruvate Kinase on kobs for the Onset of Inhibition-- Inhibition of the ATPase reaction by NaF and poly(rU) occurred during the hydrolysis of ATP to ADP (Fig. 1A). Even though enzymatically generated ADP was phosphorylated to ATP by the coupling enzymes in the reaction, a finite steady-state concentration of free ADP existed during the inhibition reaction. The steady-state concentration of free ADP should be inversely related to the concentration of pyruvate kinase. If free ADP were the reactive species in the inhibitory reaction, the concentration of ADP and consequently the value of kobs should be inversely related to the concentration of pyruvate kinase. However, the value of kobs for inhibition of the ATPase activity with 100 µg/ml poly(rU), 2.0 mM ATP, and 10 mM NaF was not affected significantly by a 32-fold change in pyruvate kinase concentration (Fig. 3). These results suggested that enzymatically generated free ADP was not the active species in the inhibitory process. Furthermore, increasing the ATP concentration from 125 to 2000 µM (Km ~ 200 µM) resulted in only a 30% decrease in kobs. These data suggested that inhibition of the enzyme by NaF during hydrolysis of ATP was the result of trapping the enzyme-bound ADP by Mg2+ and F- prior to the release of ADP and was not the result of a ternary complex of free ADP, Mg2+, and F- binding to the ATP site.


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Fig. 3.   Effect of pyruvate kinase concentration on the pseudo first-order rate constant for onset of inhibition by NaF. The time courses for inhibition of ATPase activity were determined with 100 µg/ml poly(rU) 10 mM NaF, 2.0 mM ATP, and 2.4 nM helicase in the standard assay (Method Ia). The concentration of pyruvate kinase was varied. Equation 4 was fitted to each time course to obtain a value for the pseudo first-order rate constant for onset of inhibition (kobs). The solid line was calculated by Equation 4 with a = (1.5 ± 0.3) × 10-5 s-1 unit-1 and b = (6.2 ± 0.2) × 10-3 s-1.

Titration of ATPase Activity with ADP-- The ATPase activity of a 5 µM enzyme solution was inhibited >95% after treatment for 20 min at 21 °C with 55 µM ADP, 33 mM NaF, and 133 µg/ml poly(rU) in the standard buffer that contained 3.5 mM MgCl2. Because the recovery of ATPase activity by inhibited enzyme was slow (t1/2 ~ 20 min; see below), the extent of slow reversible inhibition could be determined from the initial velocity after dilution (1:100) of the treated enzyme sample into the standard assay (Method Ia). The ATPase activity of a 5.4 µM solution of helicase treated with 33 mM NaF, 160 µg/ml poly(rU), and varying concentrations of ADP was determined. Time courses of selected inhibition reactions established that 20 min at 21 °C was sufficient time for establishing equilibrium. The ATPase activity was normalized to that of untreated enzyme or enzyme treated with all of the components except NaF (Fig. 4). Equations 5 and 6 were fitted to these data to give Delta Ainfinity  = 0.98 ± 0.03, K = 0.2 ± 0.2 µM, and n = 2.2 ± 0.1 (open circles). The stoichiometry between enzyme and ADP was firmly established by these data, whereas the value of the apparent dissociation constant (K) had significant error associated with it because the enzyme concentration was much greater than the value of K. If the titration was repeated in the absence of poly(rU) (Fig. 4, closed circles), the treated enzyme solution lost ATPase activity when assayed by Method Ia (enzyme diluted into a poly(rU)-containing assay). However, if assay Method Ib (enzyme diluted into an assay without poly(rU) followed by initiation of the reaction with poly(rU)) was used, the treated enzyme solution retained full ATPase activity (Fig. 4, closed squares). This differential effect was not observed with enzyme that had been treated with NaF, ADP, and poly(rU), but was only observed for enzyme that was treated with NaF and ADP in the absence of poly(rU).


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Fig. 4.   Titration of ATPase activity by ADP in the presence of NaF. ATPase activity was titrated in the presence (open symbols) or absence (closed symbols) of 160 µg/ml poly(rU). Helicase (5.4 µM) was incubated with 160 µg/ml poly(rU), 33 mM NaF, and the indicated total concentrations of ADP for 20 min at 21 °C. The treated enzyme solution was assayed by Method Ia (1:100 dilution). Similar values were observed with assay Method Ib. The activity was normalized to the activity of untreated enzyme. The solid line through these data (open symbols) was calculated with Equations 5 and 6 with Delta Ainfinity  = 0.98, K = 0.2, and n = 2.2 (inset). In an analogous experiment, the enzyme was reacted with NaF and ADP in the absence of poly(rU). If treated enzyme was assayed by Method Ia (1:100 dilution), the enzyme was inhibited (middle tracing). The solid line was calculated with Equations 5 and 6 and Delta Ainfinity = 0.95 and K = 4.6 µM and n = 1. If the treated enzyme was assayed by Method Ib (1:100 dilution), the enzyme was not inhibited (upper tracing).

The poly(rU)-stimulated ATPase activity was slowly inhibited by NaF and ADP in the absence of poly(rU). For example, reaction of 140 nM helicase with 500 µM ADP and 15 mM NaF resulted in a time-dependent decrease in ATPase activity that was not dependent on exposure of the treated enzyme solution to poly(rU). Enzymatic activity was determined in the standard assay by initiating the reaction with 100 µg/ml poly(rU) (Method Ib). Initial velocity data were normalized to the activity of untreated enzyme. Equation 7 was fitted to these data to give Delta Af = 0.110 ± 0.008 and k = 0.0179 ± 0.0005 min-1. This corresponded to a t1/2 for inhibition of 40 min. For comparison, inhibition of the ATPase activity under these conditions in the presence of excess poly(rU) had a t1/2 that was <10 s.

Selectivity of the Inhibition Reaction for Nucleotide Diphosphate-- NaF and poly(rU) inhibited the ATPase activity of HCV helicase in the presence of nucleotide diphosphates other than ADP. For example, incubation of 3.3 µM helicase with 200 µg/ml poly(rU), 25 mM NaF, and 10 µM CDP, GDP, UDP, and 2'-deoxyADP for 20 min at 21 °C resulted in 90.3%, 40.7%, 92.4%, and 87.8% inhibition relative to ADP, respectively, when assayed by Method 1b. Pyrophosphate did not substitute for these nucleoside diphosphates in the inhibition reaction.

Titration of ATPase Activity with NaF or MgCl2-- The ATPase activity of HCV helicase was titrated with NaF at fixed concentrations of MgCl2 (3.5 mM), ADP (40 µM), and poly(rU) (160 µg/ml). The solution was equilibrated for 20 min at 21 °C before the ATPase activity was determined by Method Ia. The activity data were normalized to that of untreated enzyme (Fig. 5A). Equation 8 was fitted to these data with Delta Ainfinity  = 0.95 ± 0.02, K = 4.3 ± 0.2 mM, and n = 2.8 ± 0.3. Similarly, the ATPase activity was titrated with MgCl2 for fixed concentrations of NaF (20 mM), ADP (40 µM) and poly(rU) (160 µg/ml). Equation 8 was fitted to these data with Delta Ainfinity  = 0.89 ± 0.03, K = 1.05 ± 0.05 mM, and n = 2.1 ± 0.2 (Fig. 5B).


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Fig. 5.   Titration of ATPase activity by NaF or MgCl2. ATPase activity was titrated by NaF or MgCl2 at fixed concentrations of poly(rU) (160 µg/ml) and ADP (40 µM). The enzyme solutions were reacted for 20 min prior to determining the ATPase activity by Method Ia. A, titration of the ATPase activity by NaF at a fixed concentration of MgCl2 (3.5 mM). The solid line was calculated by Equation 8 with Delta Ainfinity  = 0.95, K = 4.3 mM, and n = 2.8. B, titration of the ATPase activity by MgCl2 at a fixed concentration of NaF (20 mM). The solid line was calculated by Equation 8 with Delta Ainfinity  = 0.89, K = 1.05 mM, and n = 2.1.

Comparison of the Poly(rU)-stimulated ATPase Activity and the Intrinsic ATPase Activity of Enzyme Partially Inhibited by NaF, ADP, and Poly(rU)-- Helicase has a poly(rU)-stimulated ATPase activity and an intrinsic ATPase activity in the absence of poly(rU). The extent of inhibition of these activities was determined for enzyme that had been partially inhibited by 30 mM NaF, 70 µM ADP, and 5 µg/ml poly(rU). Helicase (4.6 µM) was treated with the combination of poly(rU), NaF, and ADP or with poly(rU) alone for 20 min at 21 °C. The stimulated and intrinsic activities of the treated enzyme was determined after 100-fold dilution into the standard assay in the presence or absence of poly(rU). The concentration of poly(rU) introduced into the assay (~0.2 µM base) with the treated enzyme was not sufficient to cause significant stimulation of the ATPase activity (Km ~ 20 µM). The poly(rU)-stimulated ATPase activity of enzyme treated with the combination of reagents versus enzyme treated with poly(rU) alone was reduced by 89%, whereas intrinsic ATPase activity determined in the absence of poly(rU) was only reduced by 67%.

Titration of ATPase Activity by (dU)18-- Poly(rU) was required for efficient inhibition of helicase by ADP and NaF. Because of the heterogeneous nature of poly(rU), determination of an interpretable stoichiometry between enzyme and poly(rU) in the inhibition reaction was not feasible. However, (dU)18 was active in the inhibition process and, in contrast to poly(rU), forms a 1 to 1 complex with free enzyme (12). Helicase (4.9 µM) was incubated with 30 mM NaF, 60 µM ADP and selected concentrations of (dU)18 (0 to 20 µM) for 20 min at room temperature. Enzymatic activity was determined in the standard assay by initiating the reaction with 100 µg/ml poly(rU) (Method Ib). The ATPase activity was normalized to that for untreated enzyme. ATPase activity decreased linearly as the concentration of (dU)18 was increased. Equation 9 was fitted to the data to yield a minimal (dU)18 concentration of 6.0 ± 0.2 µM (dU)18 for maximal inhibition. This corresponded to a stoichiometry of 1.2 mol of (dU)18/mol of enzyme inhibited.

First-order Rate Constant for Recovery of ATPase Activity from Inhibited Enzyme-- Reversibility of the inhibition reaction was established by monitoring the increase in ATPase activity after dilution of inhibited enzyme into the assay (Method Ia). The enzyme was inhibited by reacting 4.6 µM enzyme in the standard buffer with 200 µg/ml poly(rU), 70 µM ADP, and 30 mM NaF for 20 min at 21 °C. The inhibited enzyme was diluted 2000-fold into the standard assay buffer (Fig. 6). Equation 1 was fitted to the time course to give Vf = 0.189 ± 0.007 µM-1 s-1, Vi = (8.2 ± 0.1) × 10-3 µM-1 s-1, and kobs = (4.9 ± 0.3) × 10-4 s-1. For untreated enzyme, the value of Vf = 0.1751 ± 0.0003 µM-1 s-1.


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Fig. 6.   Time course for recovery of ATPase activity. The treated enzyme (4.6 µM) was inhibited by reaction of 4.6 µM enzyme with 30 mM NaF, 70 µM ADP, and 200 µg/ml poly(rU) for 20 min at 21 °C. The untreated enzyme solution was 4.6 µM enzyme with 200 µg/ml poly(rU). These mixtures were diluted 1:2000 into the standard assay. The solid line describing the time course for the treated enzyme solution (open symbols) was calculated with Equation 1 and Vf = 0.189 µM-1 s-1, Vi = 0.0082 µM-1 s-1, and k = 0.00049 s-1. The solid line for the time course for the untreated enzyme solution (closed symbols) was calculated with Vf = 0.175 µM-1 s-1.

Stoichiometry of ADP in the Inhibited Enzyme Complex-- Because the recovery of ATPase activity from the inhibited enzyme complex was slow, the stoichiometry between E and ADP in the inhibited complex could be determined by size exclusion chromatography. Reaction of 4.6 µM helicase in the standard buffer with 14 µM adenosine-2,8-3H 5'-triphosphate, 30 mM NaF, and 200 µg/ml poly(rU) for 15 min at 21 °C resulted in loss of over 90% of the ATPase activity. The bound and free ADP were separated by size exclusion chromatograph on a 1-ml column of P-6 resin equilibrated in the standard buffer. In a typical experiment, 27% of the total radiolabel was eluted in the column void volume. The recovery of ATPase activity of untreated enzyme in the column void volume was 58%. In the absence of enzyme, 0.8% of the total counts was eluted in the void volume. In the absence of NaF, 1.4% of the total counts was eluted in the void volume. From these data, the stoichiometry of ADP per monomeric helicase was 1.70 after normalizing for loss of enzymatic activity in the untreated sample of enzyme. A repeat of this experiment, yielded a stoichiometric ratio of 1.64.

Order of Addition of Poly(rU) and Inhibitor Species to Helicase-- Slowly reversible inhibition of ATPase activity by ADP and NaF required the presence of a nucleic acid such as poly(rU). However, enzyme treated with ADP and NaF in the absence of poly(rU) was partially inhibited (Fig. 4) when the treated enzyme solution was diluted into an assay containing excess poly(rU) (Method Ia). If the treated enzyme was diluted into a solution without poly(rU) and equilibrated for 60 s prior to the addition of poly(rU) to the assay (Method Ib), the enzyme was not inhibited. These results suggested that enzyme was forming a labile complex with ADP, MgCl2, and NaF that could be trapped with poly(rU) or could rapidly dissociate in the absence of poly(rU). Formation of this labile complex was inhibited by ATP. Thus, preincubation of 10 µM ADP and 30 mM NaF with 0.67 µM helicase for 10 min in the absence of poly(rU) yielded 63% inhibition of the ATPase activity when measured by Method Ia (100-fold dilution of the treated enzyme into the poly(rU) containing assay). If 1.0 mM ATP was included in the preincubation mixture, the enzyme was only 14% inhibited.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The combination of F- and ADP and Mg2+ in the presence of nucleic acid (poly(rU) was a slowly time-dependent inhibitor (t1/2 for dissociation was 23 min-1) of the HCV associated-ATPase activity. The combination of nucleotide diphosphates, F-, and multivalent cations such as Al3+ and Be2+ modulates the activity of numerous phosphotransferases (13-16). In many cases, Mg2+ can substitute for Al3+ and Be2+ (17-23). For example, transducin is potently activated by GDP, fluoride, and Mg2+ (23). This activated form of the enzyme was rapidly deactivated upon dilution (t1/2 ~ 5 s), whereas enzyme activated in the presence of Al3+ or Be2+ was deactivated slowly (t1/2 > 500 s). In contrast to transducin, sarcoplasmic reticulum CaATPase and the Na,K-ATPase are potently inhibited by MgFxx-2 complexes (17, 19 - 22). MgFxx-2 complexes were slowly time-dependent inhibitors that formed an inhibited enzyme species that reactivated slowly upon dilution (t1/2 = 6 min (1 mM Ca2+) and t1/2 = 3 min (150 mM NaCl), respectively). The stable complex between Mg2+ and F- with transducin and GDP has been suggested to be MgF3-1, based upon the Hill coefficient for activation (23), whereas the analogous complex with Na,K-ATPase and ADP has been suggested to be MgF2 based upon the dependence of the rate constant for onset of inhibition (11). In these complexes, MgFxx-2 is proposed to bind at the gamma -phosphate binding site for the nucleotide triphosphate (23).

HCV helicase has an intrinsic ATPase activity and nucleic acid-dependent ATPase activity. The intrinsic activity was not the result of a small amount of contaminating protein with high ATPase activity (12). However, the intrinsic ATPase activity (kcat = 3.3 s-1) and the nucleic acid-stimulated ATPase activity (kcat = 80 s-1) may be associated with different sites on the enzyme. Both of these activities were inhibited by treatment of the enzyme with F-, ADP, and poly(rU) to yield an inhibited form of the enzyme that slowly regained activity upon dilution (t1/2 = 23 min). Because Al3+ did not increase the rate of inhibition and deferoxamine, a chelator for Al3+, did not decrease the rate of inhibition, contaminating Al3+ was not involved in the inhibition process. The two ATPase activities in a sample of partially inhibited enzyme were not inhibited to the same extent (89% versus 67%). Furthermore, the stoichiometry for inhibition of the ATPase reaction indicated that 2 mol of ADP and 1 mol of nucleic acid were in the inhibited enzyme complex. The Hill coefficients for the titration of ATPase activity with F- and Mg2+ were greater than 1. This result suggested that multiple Mg2+ and F- were involved in formation of the inhibited enzyme complex. If the Hill coefficients could be equated to the number of moles of F- and Mg2+ per mol of enzyme in the complex, then 2 mol of Mg2+ and 3 mol of F- were involved in formation of 1 mol of inhibited enzyme. These results suggested two different sites on HCV helicase with ATPase activity.

The effect of ATP on the rate constant for onset of inhibition of HCV helicase by NaF and the effect of pyruvate kinase on this process suggested that MgFxx-2 trapped an enzyme-bound ADP prior to its release during the catalytic cycle and that the inhibition process was not the result of the reaction of free ADP and NaF with the enzyme. Nonetheless, ADP and F- also inhibited the ATPase activity in the presence of poly(rU) and in the absence of ATP hydrolysis. An analogous result was recently observed by Berdis and Benkovic (27) for the inhibition of ATP hydrolysis by T4 44/62 protein in the presence of Al3+ and F-. Inhibition was observed in the presence of the pyruvate kinase/lactate dehydrogenase coupling system, which rapidly phosphorylated any free ADP in solution. It was proposed that AlF4- was inhibiting the reaction by trapping 44/62·ADP prior to dissociation of the complex. This was analogous to the mechanism proposed here for the inhibition of HCV helicase associated ATPase during ATP hydrolysis in the presence of poly(rU).

The studies described herein have measured the stoichiometry between ADP and HCV helicase monomer for inhibition of the ATPase activity (n = 2.2) and for ADP binding (n = 1.7) in the presence of F- and poly(rU), and Mg2+. It was assumed that ADP and MgFxx-2 form a complex at the ATP binding site, such that the stoichiometry for ADP in the inhibited enzyme complex represented the stoichiometry for ATP binding to the enzyme. The finding that ATP protected the enzyme against inhibition by ADP and MgFxx-2 was consistent with this assumption. The major source of error in the determination of the stoichiometry between ADP and monomeric HCV helicase was estimation of active protein concentration. The error in the concentration of ADP used for titration of the enzyme was minimized by spectrophotometrically determining the concentration of ADP in each solution. In these studies protein concentration was calculated with an extinction coefficient based upon (rU)15 binding sites (epsilon 280 = 88 mM-1 cm-1(12)). Thus, the stoichiometry reported herein was actually the measured stoichiometry between ADP and DNA binding sites. The stoichiometry of (dU)18 binding to enzyme monomer in the inhibited enzyme complex was 1.2, which was consistent with the value of this extinction coefficient.

The suggestion that HCV helicase has two ATP binding sites per monomer (DNA binding site) could have significant implications for the mechanism of helicase activity, such as the requirement of for the coupling of hydrolysis of ATP at two sites during the unwinding of duplex DNA. The interaction of ATP with other single-stranded nucleic acid-dependent ATPase appears to be a multistep process. For example, the first cycle of interaction of ATP with by E. coli recA was too slow to be on the catalytic pathway for ATP hydrolysis (28). Wong and Lohman (6) have suggested from studies on the K28I mutant of Rep helicase that a global conformational change of the protein occurs prior to the first turnover. Possibly, these results are suggesting multiple binding sites for ATP on these enzymes as well.

    ACKNOWLEDGEMENTS

We gratefully acknowledge S. Short and E. Furfine for helpful discussions during the course of this work.

    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; ATPase, ATPase activity associated with the HCV NS3 helicase domain encompassing amino acids 1193-1657 of the HCV type 1b polyprotein; MOPS, 3-(N-morpholino)propanesulfonic acid; E, free enzyme in the presence of Mg2+; E·ADP, binary complex between E and ADP; E·ADP·F, ternary complex among E, ADP, and F- without specifying the stoichiometry.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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