 |
INTRODUCTION |
Even though helicase activity was identified and the associated
protein was purified over 20 years ago (1), the kinetic and chemical
mechanisms for this class of enzymes have not been elucidated. In
particular, the mechanism of coupling ATP hydrolysis to the unwinding
of double-stranded DNA is incompletely understood. From extensive
studies on the mechanism of action of Escherichia coli Rep
helicase, Lohman and co-workers (2, 3-12) have proposed that the
catalytically active species of this enzyme is a DNA stabilized dimer.
The two DNA-binding sites in the catalytic dimer unwind defined lengths
of the DNA duplex by alternatively binding duplex and single-stranded
DNA in a process coupled to ATP hydrolysis (2, 5, 6, 12). Recently, it
has been shown that E. coli helicase II unwinds
approximately 4 base pairs during each catalytic cycle (13). Nucleotide
binding also regulates the binding of single-stranded and
double-stranded DNA to E. coli Rep helicase. In particular,
ATP stimulates the rate of single-stranded DNA exchange by increasing
the rate constant for dissociation of DNA (10). Because the active form
of this enzyme is a dimer (2, 3, 11), there are minimally two potential
sites for ATP binding. Bjornsen et al. (10) have provided
kinetic evidence that suggested these two sites communicate.
Recently Wong and Lohman (14) made heterodimers of Rep
helicase containing ADP-AlF4 tightly bound at the ATP site
and covalently cross-linked single-stranded DNA at the DNA-binding
site. Through the use of cis- (substrate analogues on the
same subunit) and trans (substrate analogues on different
subunits)-labeled dimers, they concluded that most of the observed
ATPase activity was the result of the cis-labeled subunit.
The ATPase activity of the subunit lacking single-stranded DNA was
small because of a slow conformational change occurring prior to ADP
release and not due to a diminished rate of ATP hydrolysis (14). Even
though the kinetic mechanism for ATP hydrolysis by Rep helicase in the
presence of single-stranded DNA has been clearly defined, the chemical
mechanism for coupling ATP hydrolysis to double-stranded DNA unwinding
is still unclear.
Hepatitis C virus (HCV)1
genome encodes for an RNA helicase that presumably is essential for
viral replication (15). Recently, a crystal structure of the HCV
helicase has been reported (16). However, 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, and in
the absence of nucleic acid or nucleotide. Crystal structures of Rep
helicase from E. coli (17) and a helicase from
Bacillus stearothermophilus have also been reported.
Co-crystals of Rep helicase with nucleic acid and ADP in the absence of
Mg2+ implicated motifs Ia, V, and III for single-stranded
DNA binding, motifs I and IV for nucleotide binding, and motifs II and
IV possibly functioned in the coupling of nucleotide and
single-stranded DNA binding (17). These structures suggested a single
nucleotide-binding site per monomer.
HCV helicase has a large intrinsic ATPase activity
(kcat = 3 s
1 (19)).
Single-stranded DNA or RNA increases the kcat
value up to 30-fold (19). In contrast, the kcat
values for ATPase activities of bacterial helicases such as Rep
helicase and helicase II are increased over 1000-fold by
single-stranded DNA (11). The turnover numbers for ATP hydrolysis by
HCV helicase and Rep helicase in the presence of nucleic acid are
similar. 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 suggested
that either HCV helicase ATPase activity was associated with a single
site that was not tightly coupled to nucleic acid binding or HCV
helicase may have ATPase activity associated with two separate sites on
the enzyme. Further support for the latter suggestion was the finding
that stoichiometry for ADP binding to the enzyme in the presence of excess Mg2+, F
, and poly(rU) was two
nucleotides per nucleic acid-binding site (30).
HCV helicase forms spectrofluorometrically detectable intermediates
with DNA (19) that react with ATP. The kinetic competence of the
intermediates formed from the reaction of E·DNA with ATP or "E·ATP" with DNA was evaluated from
pre-steady-state and steady-state data. In summary, the intermediates
associated with these fluorescent changes were catalytically
incompetent. These results suggested either the fluorescent changes
were monitoring a catalytically inactive ATP-binding site with a
fluorescently silent catalytically active site or the first cycle of
interaction of enzyme with ATP was unique relative to subsequent cycles
of ATP hydrolysis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Rabbit muscle pyruvate kinase, rabbit muscle
lactate dehydrogenase, NADH, ATP, phosphoenolpyruvate, and MOPS were
from Sigma. HF16 and the 16-mer from Oligo Therapeutics, Inc., were
purified by electrophoresis through 20% polyacrylamide gels in 6 M urea. Oligomer concentrations were calculated from the
absorbance at 260 nm and the extinction coefficients provided by Oligo
Therapeutics. The standard buffer was 0.5 M MOPS
K+ and 3.5 mM MgCl2 at pH 7.0. HCV
helicase was purified as described previously (19). The concentration
of monomer was calculated with
280 = 88 mM
1 cm
1 (19).
Assay of ATPase Activity--
ATPase was assayed
spectrophotometically by the absorbance change at 340 nm resulting from
the oxidation of NADH that was coupled to the phosphorylation of ADP
through pyruvate kinase and lactate dehydrogenase (20). Rates were
calculated using a 
340 = 6.22 mM
1 cm
1. A solution of the
coupling components (40 units/ml pyruvate kinase, 40 units of lactate
dehydrogenase, 8.0 mM phosphoenolpyruvate, and 800 µM NADH) was prepared in the standard buffer at 5 °C. This solution was diluted 4-fold with the standard buffer containing the selected concentrations of DNA and ATP. The reactions were initiated with HCV helicase. The t1/2 for
phosphorylation of 5 µM ADP in this assay was 3 s.
The measured rate of ATP hydrolysis was not affected by decreasing the
standard concentration of the coupling enzymes NADH and
phosphoenolpyruvate by 50%.
Titration of HCV Helicase--
Titration of helicase
(E) with a ligand (L) was monitored by quenching of
intrinsic protein fluorescence or by quenching of fluorescence of
hexachlorofluoresceinyl-labeled DNA upon formation of the enzyme-ligand
complex (E·L). It was assumed that this process could be
described by the simplified mechanism of Equation 1, where K
was the dissociation constant of E for L,
[Et] was the concentration of enzyme sites binding
ligand, and [Lt] was the concentration of ligand.
|
(Eq. 1)
|
Furthermore, it was assumed that the fractional fluorescence
(F([L]t)) remaining was related to the total
concentration of added ligand sites ([Lt]) by Equation 2 in
which
F
was the fractional fluorescence
decrease resulting from conversion of E to
E·L.
|
(Eq. 2)
|
Because the concentration of enzyme was in many cases comparable
to the dissociation constant of the enzyme for L, the concentration of
E·L for the simple scheme of Equation 1 was related to
[Lt] by Equation 3.
|
(Eq. 3)
|
During the titration of E with the 16-mer, which was
monitored by the quenching of the intrinsic protein fluorescence
(
ex = 280 nm and
em = 340 nm), there was
significant drift in the fluorescence signal. This was corrected for by
a blank titration in which the experimental signal obtained after
addition of the 16-mer to the enzyme was normalized to that observed
after addition of an equivalent volume of buffer to the enzyme. The
time between additions of sample to the protein was 30 s.
Steady-state Kinetic Data Analysis--
The dependence of the
rate of ATP hydrolysis by HCV helicase on ATP, HF16, and the 16-mer
concentrations was complicated. Equation 4 for a random rapid
equilibrium scheme in which two molecules of ATP and one molecule of
DNA bound to helicase described these data. The parameter of interest
for these studies was kcat, which was relatively
independent of the model fitted to the data. The substrate coefficients
in Equation 4 were not used in the present studies.
|
(Eq. 4)
|
Analysis of Time Courses--
The time courses for fluorescence
changes were described by exponential functions (Equation 5).
|
(Eq. 5)
|
Where F(t) was the observed signal at time
equal to t, Fi was the respective
amplitude of the phase associated with ki.
F0 was the final value of the fluorescence. The
reaction of ligand (L) with enzyme was described by the two-step
mechanism of Equation 6. For this mechanism, the dependence of the
observed rate constant (kobs) on ligand
concentration ([L]) was given by Equation 7.
|
(Eq. 6)
|
|
(Eq. 7)
|
|
(Eq. 8)
|
Where kobs([L]) was the value of
kobs for the indicated concentration of L;
k
2 was the first-order rate constant at zero
concentration of L; the sum of k2 and
k
2 was the first-order rate constant at
infinite ligand concentration; and K was the concentration
of L such that kobs = k
2 + 0.5k2.
F([L]) was the amplitude of the fluorescence change as
a function of [L] and
F
was the maximal
signal change. Equations 7 and 8 were fitted to the amplitude data and
the kinetic data globally to yield estimates for K,
K
2, k2, and
F
. If the value of K was much
greater than the concentrations of L, Equation 7 was approximated by
Equation 9.
|
(Eq. 9)
|
If L were a substrate for the enzyme, Equation 6 was modified to
include an additional step (kcat) for the return
of EL to E. In this modified scheme, the
k
2 term in Equations 7-9 would be the sum of
k
2 and kcat
(k
2,obs).
General Methods--
Fluorescence spectra were recorded on a
Kontron SFM 25 spectrofluorometer. Intrinsic protein fluorescence data
(
ex = 280-300 nm, tryptophanyl and tyrosinyl residues,
em = 340 nm) were corrected for inner and outer filter
effects. Fluorescence of HF16 was monitored with
ex = 510 nm and
em = 550 nm. Absorbance and conventional kinetic data were obtained from a UVIKON 860 spectrophotometer at
25 °C or from a Spectramax 250 plate reader at 21 °C. Rapid reactions were monitored on an Applied Photophysics SX.17MV
Spectrophotometer (Leatherhead, UK). All concentrations of reactants
are given after mixing on the stopped-flow spectrofluorometer. Entrance
and exit slits were 10 mm in the absorbance mode and 2 mm in the
fluorescence mode. The fluorescence of the hexachlorofluoresceinyl
moiety was monitored on the stopped-flow spectrofluorometer with
ex = 500 nm and
ex >530 nm. Intrinsic
protein fluorescence was monitored with
ex = 280-290 nm
and
em >305 nm. Stopped-flow time courses were an
average of 4-6 experiments. Fluorescence data from the stopped-flow
spectrophotometer were presented as the voltage change from the
beginning of the reaction. These changes were not normalized to the
initial fluorescence of the solution because the solutions contained
varying amounts of HF16 that were in excess of enzyme. For a group of
experiments associated with a particular figure, the photomultiplier
voltage was held constant. However, the magnitude of the voltage
changes should not be compared between groups of experiments, only the
voltage changes associated with a single group of experiments should be
compared. The appropriate equations were fitted to the data by
nonlinear least squares using SigmaPlot from Jandel Scientific (Corte
Madera, CA).
 |
RESULTS |
kcat for ATP Hydrolysis by HCV Helicase with HF16 and
the 16-mer as Single-stranded Nucleic Acid Effectors--
The goal of
the studies described herein was to determine if the formation of
spectrally observed intermediates was a step in the steady-state
catalytic cycle of ATP hydrolysis by HCV helicase. The criteria for
kinetic competence of an intermediate in the catalytic cycle was that
the value of the limiting first-order rate constant for the approach to
the steady-state was at least 4-fold greater than the
kcat for ATP
hydrolysis.2 Thus, the value
of kcat was the number of
importance determined from steady-state rate data. The value for
kcat for ATP hydrolysis by HCV helicase is
dependent on the single-stranded DNA effector. For example
(dU)18 increased the value of kcat
30-fold (19). With HF16 as the DNA effector, the ATPase activity of the
enzyme was inhibited at small ATP concentrations and enhanced at large ATP concentrations (Fig. 1). The complex
concentration dependence of the initial rate data was adequately
described by Equation 4 with kcat = 2.73 ± 0.09 s
1 for HF16. The kcat for the
16-mer was calculated to be 36 ± 7 s
1 from an
analogous data set. The rate of hydrolysis of 1.6 mM ATP in
the presence of 1.4 µM HF16 (data not shown) was linearly dependent on enzyme concentrations between 1 and 60 nM.
This result indicated that the value of kcat was
independent of enzyme concentration so that it was valid to compare
kcat values with kinetic parameters determined
at different enzyme concentrations. The values for the
Km of the enzyme for ATP at near-saturating
concentrations of HF16 (1600 nM) and the 16-mer (6000 nM) were determined to be 320 ± 50 and 410 ± 20 µM, respectively.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
Steady-state hydrolysis of ATP with HF16 as
the single-stranded DNA effector. The initial rate of ATP
hydrolysis by HCV helicase was determined for selected ATP and HF16
concentrations at 21 °C. The concentration of enzyme was 38.5 nM. The solid lines were calculated by Equation 4. The value of kcat was 2.73 s 1.
|
|
Affinity of HCV Helicase for HF16 and the 16-mer--
The binding
of HCV helicase to HF16 was monitored by the quenching of HF16
fluorescence upon formation of E·HF16. Titration of 22.4 nM HF16 with HCV helicase (Fig.
2) was described by Equations 2 and 3
(L = HF16) with K = 0.48 ± 0.02 nM and
F
= 0.457 ± 0.006. Quenching of intrinsic protein fluorescence monitored the
binding of HCV helicase to the 16-mer. Titration of 33.5 nM E (Fig. 2, inset) was described by Equations 2 and 3 (L = 16-mer) with K = 2.2 ± 0.4 nM and
F
= 0.377 ± 0.006. Because the concentration of the fixed ligand was many fold greater than the Kd for dissociation of E·DNA, the
calculated values for Kd were associated with
considerable error. Nonetheless, these estimates for the values of
Kd were similar to the estimate values calculated
from bimolecular association rate constants and dissociation rate
constants (see below).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Titration of helicase by HF16 and the
16-mer. The binding of HF16 to enzyme was monitored by the
quenching of the fluorescence of HF16 ( ex = 510 nm and
em = 552 nm). The fluorescence of HF16 (22.4 nM) was titrated with helicase. The solid line
was calculated with Equations 2 and 3 and the following values for the
parameters: K = 0.48 nM and
F = 0.457. The binding of enzyme to the
16-mer (inset) was monitored by the quenching of the protein
fluorescence ( ex = 280 nm and em = 340 nm). The fluorescence of helicase (33.5 nM) was titrated
with the 16-mer. The solid line was calculated by Equations
2 and 3 with K = 2.2 nM and
F = 0.377.
|
|
Kinetics for Equilibration of HCV Helicase with HF16 and the
16-mer--
The time course for quenching of the fluorescence of HF16
by HCV helicase was a first-order process (Fig.
3, inset). The pseudo first-order rate constant for this process
(kobs) was linearly dependent on the
concentration of HF16 with [HF16] > [E] (Fig. 3). These
data indicated that the Kd for formation of the
initial complex between E and HF16 (Equation 6) was much
greater than the highest concentration of HF16 tested. Consequently,
Equation 9 was fitted to these data to estimate a value for the
apparent bimolecular rate constant for association of HCV helicase with HF16 (k2/Kd in Equation 9
with k
2 = 0) of 640 ± 20 µM
1 s
1. The reaction of the
16-mer with HCV helicase was monitored by the quenching of the
intrinsic protein fluorescence upon formation of E·16. The
pseudo first-order rate constant for this reaction was also linearly
dependent on the concentration of the 16-mer yielding an analogous
bimolecular rate constant of 99 ± 2 µM
1 s
1.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Kinetics of the reaction of HCV helicase with
HF16. The reaction of 13 nM helicase with selected
concentrations of HF16 was monitored on the stopped-flow
spectrofluorometer with ex = 500 nM and
em >530 nM. Inset, time course
for the reaction of 134 nM HF16 with the enzyme. The
solid line was calculated with Equation 5 (i = 1) and k1 = 80.5 s 1. The value
of kobs (k1) was linearly
dependent on HF16 concentration. The solid line was
calculated by Equation 9 with
k2/Kd = 640 µM 1 s 1.
|
|
The dependence of the pseudo first-order rate constant for the reaction
of HF16 with HCV helicase appeared to extrapolate to a small value
(Fig. 3, ordinate intercept) at zero concentration of HF16.
This result suggested that the rate constant describing the
dissociation of E·HF16 (k
2 in
Equation 9) was very small. A direct measure of the dissociation of
E·HF16 was made by monitoring the associated increase in
fluorescence of HF16 (
ex = 500 nm,
em
>530 nm) upon release from the enzyme in the presence of excess
dextran sulfate that trapped free HCV helicase (Equation 14
).
The fluorescence (
ex = 500 nm,
em
>530 nm) of a solution of 13.2 nM E and 18.8 nM HF16 increased in a first-order process (kobs) upon addition of 2.0 mM
dextran sulfate. kobs had a value of 0.087 ± 0.001 s
1 with 2.0 mM dextran sulfate and a
similar value (0.109 ± 0.001 s
1) with 0.4 mM dextran sulfate. Because kobs was
similar for two dextran sulfate concentrations, this value was assigned
to the dissociation rate constant (k
2) for
E·HF16. Similar experiments with E·16
(
ex = 280 nm,
em >305 nm) yielded a
value for k
2 of 0.096 ± 0.009 s
1.
The apparent dissociation constants (K) of E for
HF16 and the 16-mer were calculated from the relationship
K ~ Kdk
2/k2, where k
2 was the effective rate constant for
dissociation of E·HF16, and
k2/Kd was the effective
association rate constant for reaction of HCV helicase and HF16
(Equation 6, L = HF16). The values of K for HCV
helicase and HF16 and the 16-mer were calculated from the kinetic data
to be 0.14 and 1.0 nM, respectively, which were in
reasonable agreement with the values estimated from titration data
(0.48 and 2.2 nM, respectively).
Kinetics for the Approach to the Steady-state from E·ATP and HF16
or the 16-mer--
The time courses for the approach to the
steady-state starting with 2.0 mM ATP, 13.2 nM
E·ATP, and selected concentrations of HF16 were
first-order processes (kobs). The dependence of
kobs on HF16 concentration was described by
Equations 7 and 8 with k
2,obs = 0.37 ± 0.02 s
1, k2 = 1.2 ± 0.2 s
1, K = 240 ± 70 nM,
and
F
= 0.40 ± 0.03 V (Fig.
4). Similarly, the approach to the
steady-state starting with 50 nM E·ATP and selected concentrations of the 16-mer was a first-order processes. Because the highest concentration of the 16-mer was significantly less
than the Kd for the reaction, Equation 9 was fitted to the data with k
2,obs = 2.0 ± 0.1 s
1, k2/Kd = 0.41 ± 0.01 µM
1 s
1.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Kinetics of the reaction of
E·ATP with HF16 in the presence of 2.0 mM
ATP. E·ATP was generated by premixing 26.4 nM
enzyme with 2.0 mM ATP. E·ATP was mixed with
selected concentrations of HF16 as described in the legend to Fig. 3.
The dependence of the observed pseudo first-order rate constant for
approach to the steady-state and the amplitudes (inset) were
described by Equations 7 and 8. The value of
kobs in the absence of HF16 was an independently
determined value for the rate constant for dissociation of
E·ATP·HF16. The solid line was calculated by
these equations with k 2 = 0.37 s 1, k2 = 1.2 s 1,
K = 240 nM, and
F = 0.4.
|
|
Dissociation of E·HF16·ATP or E·16·ATP--
The
dissociation constant of the enzyme for HF16 was increased over
100-fold by ATP (compare data of Fig. 2 with that of Fig. 4,
inset). Consequently, mixing ATP with a solution of
E·HF16 ([HF16] < [KHF16])
should result in the partial dissociation of HF16 from
E·ATP as depicted in Equation 15.
The release of HF16 from the enzyme was monitored by the
associated increase in fluorescence of HF16 (
ex = 500 nm,
em >530 nm). The approach to the steady-state upon
addition of 1.0 mM ATP to 9.3 nM
E·HF16 that was generated from 9.3 nM helicase
and 17.7 nM HF16 was a biphasic process that indicated that
ATP bound to the E·HF16 complex to form an intermediate
("E·HF16·ATP") from which HF16 dissociated (Fig.
5). Equation 5 (i = 2)
was fitted to these data to give F0 =
0.494 ± 0.003 V, F1 =
3.98 ± 0.2 V, k1 = 1.01 ± 0.03 s
1,
F2 = 4.3 ± 0.2 V, and
k2 = 0.403 ± 0.008 s
1. The
early phase of the reaction was attributed to formation of
E·HF16·ATP from E·HF16 and ATP, whereas the
late phase of the reaction was attributed to dissociation of HF16 from
E·HF16·ATP. When dextran sulfate was added to a
steady-state mixture of ATP, HCV helicase, and HF16 to trap free
E as it was cycled during catalysis, the time course for
formation of free HF16 was monophasic (Fig. 5, inset).
Equation 5 (i = 1) was fitted to these data with F0 =
0.67 ± 0.002 V,
F1 = 0.067 ± 0.002 V, and
k1 = 0.341 ± 0.002 s
1. The
value of the rate constant for the latter process was in good agreement
with the value for the late phase of the reaction in the presence of
ATP alone (0.403 s
1).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Time course for dissociation of
E·HF16 in the presence of ATP. E·HF16, which
was generated from 9.3 nM helicase and 17.7 nM
HF16, was reacted with 2.0 mM ATP. Changes in the
fluorescence of HF16 were monitored as described in the legend to Fig.
3. The solid line was calculated with Equation 5
(i = 2) and F0 = 0.494 V,
F1 = 3.98 V, k1 = 1.01 s 1, F2 = 4.3 V, and
k2 = 0.403 s 1. If
E·HF16 was incubated with 2.0 mM ATP prior to
mixing with 1 mM dextran sulfate and 2.0 mM ATP
to trap free enzyme as E·HF16 dissociated, the time course
was monophasic (inset). The solid line was
calculated by Equation 5 (i = 1) with
F0 = 0.847 V, F1 = 0.847 V, and k1 = 0.341 s 1.
|
|
ATP also induced the dissociation of E·16. The release of
the 16-mer from E·16 was monitored by the associated
change in intrinsic protein fluorescence (
ex = 280 nm,
em >305 nm) upon formation of (E·ATP) from
(E·16·ATP). The addition of 1.0 mM ATP to a
solution of 50 nM E·16 generated from 50 nM E and 75 nM 16-mer resulted in a
biphasic increase in fluorescence. These data (not shown) were
described by Equation 5 (i = 2) with
F0 =
0.129 ± 0.001 V,
F1 =
0.83 ± 0.07 V,
k1 = 2.34 ± 0.05 s
1,
F2 = 1.08 ± 0.07 V, and
k2 = 1.70 ± 0.02 s
1. The
early phase of the reaction was attributed to formation of
E·16·ATP from E·16 and ATP, whereas the
late phase of the reaction was attributed to dissociation of the 16-mer
from E·16·ATP.
Kinetics for the Approach to the Steady-state from E·HF16 or
E·16 and ATP--
The previous results indicated that the reaction
of E·HF16 with ATP to form E·HF16·ATP was
associated with a fluorescence decrease (Fig.
6A). Because the concentration
of HF16 in these experiments was less than the dissociation constant of
E·ATP for HF16, E·HF16·ATP partially
dissociated to E·ATP and HF16. The dissociation phase of
the reaction was eliminated by including high concentrations of HF16 in
the reaction. Thus, the reaction of 2.0 mM ATP with 50 nM E·HF16, which was generated by premixing 50 nM E with 492 nM HF16, was a first-order
process (Fig. 6A). Equation 5 (i = 1) was
fitted to these data with F0 = 0.262 ± 0.01 V, F1 =
0.262 ± 0.001 V, and
k1 = 1.67 ± 0.03 s
1. This
rate constant described the approach to the steady-state after mixing
E·HF16 with ATP. The values of pseudo first-order rate
constant (kobs) and the amplitudes of the
fluorescence change for this reaction were determined for selected ATP
concentrations (Fig. 6B). Equations 7 and 8 were fitted to
these data with K = 121 ± 7 µM,
k2 = 1.82 ± 0.03 s
1,
k
2,obs = 0.05 ± 0.01 s
1,
and
F
= 0.29 ± 0.01 V. If
E·ATP·HF16 were a catalytic intermediate in ATP
steady-state ATP hydrolysis as depicted in Equation 15, the value of
k
2,obs should have been the sum of
k
2 and kcat.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Time course for the reaction of
E·HF16 with ATP in the presence of excess HF16.
A, E·HF16, which was generated from 50 nM helicase and 492 nM HF16, was reacted with
2.0 mM ATP. Changes in fluorescence of HF16 were monitored
as described in the legend to Fig. 3. The solid line was
calculated with Equation 5 (i = 1) and
F0 = 0.262 V, F1 = 0.262 V, and k1 = 1.67 s 1.
B, effect of ATP concentration on the time course of the
reaction of 18.6 nM E·HF16 with ATP. The ATP
dependence of the pseudo first-order rate constant and the observed
fluorescence change (inset) were described by Equations 7
and 8. The solid lines were calculated by these equations
with k2 = 1.82 s 1
k 2 = 0.05 s 1,
K = 121 µM, and
F = 0.28.
|
|
A data set similar to that for E·ATP and HF16 was
collected for the reaction of E·ATP with the 16-mer.
E·16 was generated by premixing 50 nM
E with 12 µM 16-mer. Addition of 1.0 mM ATP resulted in a first-order increase in intrinsic
protein fluorescence (data not shown). Equation 5 (i = 1) was fitted to these data with F0 = 0.109 ± 0.002 V, F1 =
0.109 ± 0.002 V, and
k1 = 2.55 ± 0.07 s
1. The
values of pseudo first-order rate constant
(kobs) and the amplitudes of the fluorescence
change for this reaction were determined for selected ATP
concentrations. Equations 7 and 8 were fitted to these data with
K = 170 ± 20 µM,
k2 = 2.9 ± 0.2 s
1,
k
2,obs = 0.49 ± 0.06 s
1,
and
F
= 0.104 ± 0.004 V. The kinetic
parameters for these reactions are summarized in Table
I.
View this table:
[in this window]
[in a new window]
|
Table I
Kinetic parameters for the reaction of HCV helicase with selected
oligomers and ATP
Eq. 6 was fitted to the fluorescence data to obtain estimates for the
kinetic parameters.
|
|
 |
DISCUSSION |
Helicases catalyze the ATP-dependent unwinding of
double-stranded DNA. The mechanisms for the unwinding reaction are
being actively investigated in numerous laboratories (2, 18, 21-24). Recent mechanistic studies on E. coli Rep helicase are
forming the basis for a detailed understanding of the kinetic mechanism for DNA unwinding by a helicase (2). Lohman (6) has classified the
mechanism for helicase action as being either "active or
"passive." In a passive type mechanism, the enzyme binds
preferentially to single-stranded DNA and unwinds double-stranded DNA
by binding to single-stranded DNA that is formed transiently from the
duplex as the result of thermal fluctuations. In contrast, an active mechanism requires that the enzyme bind to both double-stranded DNA and
single-stranded DNA, and the enzyme destabilizes multiple base pairs
during each catalytic step. This mechanism requires that the enzyme
have multiple DNA-binding sites either on the same subunit or as an
oligomeric protein. During catalysis, one DNA-binding site
alternatively binds double-stranded and single-stranded DNA as the
enzyme rolls along the double-stranded DNA. Whichever mechanism is
operative for unwinding of double-stranded DNA, the coupling of ATP
hydrolysis to translocation is crucial for the unwinding process.
The coupling of ATP hydrolysis to conformational transitions of other
domains in members of helicase superfamily I is being investigated by
site-directed mutagenesis of conserved amino acids in each of the six
motifs. For example, E. coli helicase II mutants D248N,
K35M, and R284A greatly reduced the single-stranded DNA-stimulated ATPase activity (25-27). Recently, site-specific substitutions in each
of the six motifs of HSV helicase (superfamily 1) produced large
effects when in motifs I and II and small effects when in motifs
III-VI on DNA-dependent ATPase activity (28). To
supplement these studies, our goal was to characterize the
single-stranded DNA-stimulated ATPase activity of HCV helicase by
steady-state and pre-steady-state kinetic techniques. In particular,
the kinetic competence of spectrofluorometrically observed
intermediates was determined. HCV helicase quenched the fluorescence of
HF16, whereas HF16 and the 16-mer quenched the intrinsic protein
fluorescence. The kinetics for formation of these intermediates with
modified fluorescence properties were compared with the values for
kcat for ATP hydrolysis to decide whether or not
the intermediates were kinetically competent for catalysis of ATP
hydrolysis.
The dissociation of DNA from the enzyme in the presence of ATP was too
slow for this process to be an obligatory step in the catalytic cycle.
The rate constants for dissociation of E·HF16 and
E·16 in the presence of 2.0 mM ATP were 0.341 s
1 and 1.70 s
1, respectively, whereas the
values of kcat were 2.73 and 36 s
1, respectively. Because DNA dissociated from
E·DNA infrequently during the catalytic cycle, the enzyme
cycled primarily between E·DNA and
E·DNA·ATP during ATP hydrolysis. If
E·DNA·ATP were on the pathway for hydrolysis of ATP, the
pseudo first-order rate constant for the approach to the steady-state
starting with E·DNA and a saturating concentration of ATP
must be at least 4-fold greater than the value of
kcat.2 Furthermore, the
concentration of ATP that resulted in 50% of the maximal amount of
this intermediate must be greater than or equal to the
Km for ATP. These conditions were not met by the
pre-steady-state intermediate formed from E·DNA and ATP. For instance, the maximal value for the first-order rate constant for
the approach to the steady-state concentration of
E·HF16·ATP starting with E·HF16 and ATP was
1.82 s
1, whereas kcat was 2.73 s
1. The minimal value for the rate constant for approach
to the steady state at saturating concentration of ATP has to be 10.9 s
1 for this process to be on the catalytic cycle.
Furthermore, the concentration of ATP that yielded 50% of the maximal
amount of the intermediate for a saturating concentration of HF16
should be equal to the Km for the steady-state rate
of ATP hydrolysis. The value of Km for the ATP
at saturating concentrations of HF16 was 320 µM, whereas
the concentration of ATP that yield 50% of the intermediate was
only 3 µM. Thus, formation of this intermediate was
not a competent step in the catalytic cycle of ATP hydrolysis.
These results suggested either that the formation of
E·DNA·ATP occurred independently of and was unrelated to
the catalytic process or that formation of this species introduced the
active form of the enzyme into the catalytic cycle. If the latter were the case, an isomerization step could be included in the kinetic mechanism such that reaction of enzyme with the first molecule of ATP
was different than its reaction with subsequent molecules of ATP
(Scheme I). In this model, steady-state
ATP hydrolysis was mediated principally through cycling of
HF16 (Scheme I), whereas the reaction monitored on
the stopped-flow spectrofluorometer was conversion of EHF16 to
HF16. These data have not addressed the fate of ATP in
the conversion of EHF16 to
HF16.
Possibly a structural isomerization dependent on phosphorylation of the
enzyme was involved that transformed inactive enzyme into a
catalytically active form. If this were the case, formation of
HF16 and its reversion to EHF16
(<0.05 s
1 in Scheme I) could involve net hydrolysis of
ATP.
The data presented herein for hydrolysis of ATP by E·HF31
suggested that the first cycle of interaction of ATP with
E·HF31 was unique relative to subsequent cycles of
interaction of the enzyme with ATP. This concept has been suggested for
other DNA unwinding proteins such as the E. coli Rep
helicase and the E. coli Rec A protein. For Rep helicase,
the approach to the steady state for the reaction of 1.0 mM
ATP with enzyme preloaded with 2-aminopurine-labeled
dT(pT)15 appeared to be too slow for the formation of
ternary complex to be a kinetically competent step during ATP
hydrolysis (10). Furthermore, Wong and Lohman (14) have suggested from
studies on the K28I mutant of Rep helicase that a global conformation
change of the protein occurs prior to the first turnover. Similar
results have been described for a mutant E. coli Rec A
protein (H163W) that catalyzed single-stranded DNA-dependent ATPase. The fluorescence of the H163W protein
was used to monitor conformational changes occurring during ATP
hydrolysis (29). The first cycle of interaction of the mutant protein
with ATP was too slow to be on the catalytic pathway. Consequently, Stole and Bryant (29) proposed an isomerization model similar to
that described in Scheme I. The steady-state hydrolysis of ATP by these
DNA unwinding proteins was preceded by a unique isomerization of the
initial ternary enzyme-DNA-ATP complex that may be a general phenomenon
for all DNA unwinding proteins.
I thank S. Short, F. Preugschat, and E. Furfine for helpful discussions during the course of this work.
At saturating substrate concentration, the pseudo first-order rate
constant for approach to the steady-state (kobs)
and kcat are given by Equations 11 and
12.