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 |
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 |
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
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
260 = 9.35 mM
1 cm
1 and
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
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
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
Vf .
|
(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.
|
(Eq. 2)
|
|
(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.
|
(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.
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
A
was the fractional
activity decrease resulting from conversion of E to
E·ADP.
|
(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.
|
(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).
|
(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
A
was the maximal
decrease in activity at an infinite concentration of L.
|
(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]).
|
(Eq. 9)
|
|
(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 |
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.
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|
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.
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|
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.
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|
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
A
= 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 A = 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
A = 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).
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|
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
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
A
= 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
A
= 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 A = 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 A = 0.89, K = 1.05 mM, and n = 2.1.
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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 |
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
-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
(
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.
We gratefully acknowledge S. Short and
E. Furfine for helpful discussions during the course of
this work.