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Inhibition of the Hepatitis C Virus Helicase-associated ATPase Activity by the Combination of ADP, NaF, MgCl2, and Poly(rU)

1998; Elsevier BV; Volume: 273; Issue: 13 Linguagem: Inglês

10.1074/jbc.273.13.7390

1083-351X

David Porter,

Pneumocystis jirovecii pneumonia detection and treatment

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 (t½ = 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 (t½ > 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. 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 (t½ = 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 (t½ > 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. Hepatitis C virus (HCV) 1The 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. 1The 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.genome encodes for an RNA helicase that presumably is essential for viral replication (1Kim D.W. Gwack Y. Han J.H. Choe J. Biochem. Biophys. Res. Commun. 1995; 215: 160-166Crossref PubMed Scopus (289) Google Scholar). Helicases catalyze the separation of double-stranded nucleic acids into single-stranded nucleic acids with the concomitant hydrolysis of ATP (2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (670) Google Scholar). The mechanism of coupling of ATP hydrolysis to double-stranded nucleic acid unwinding is unclear (2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (670) Google Scholar, 3Moore K.J.M. Lohman T.M. Biophys. J. 1995; 68: 180s-185sAbstract Full Text PDF PubMed Scopus (18) Google Scholar).Escherichia coli Rep helicase is the most thoroughly characterized helicase in terms of characterizing the binding of DNA and the associated ATPase activities (3Moore K.J.M. Lohman T.M. Biophys. J. 1995; 68: 180s-185sAbstract Full Text PDF PubMed Scopus (18) Google Scholar, 4Moore K.J.M. Lohman T.M. Biochemistry. 1994; 33: 14550-14564Crossref PubMed Scopus (60) Google Scholar, 5Bjornson K.P. Wong I. Lohman T.M. J. Mol. Biol. 1996; 263: 411-422Crossref PubMed Scopus (28) Google Scholar, 6Wong I. Lohman T.M. Biochemistry. 1997; 36: 3115-3125Crossref PubMed Scopus (25) Google Scholar, 7Bjornson K.P. Moore K.J.M. Lohman T.M. Biochemistry. 1996; 35: 2268-2282Crossref PubMed Scopus (47) Google Scholar, 8Wong I. Moore K.J.M. Bjornson K.P. Hsieh J. Lohman T.M. Biochemistry. 1996; 35: 5726-5734Crossref PubMed Scopus (37) Google Scholar). 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 (9Korolev S. Hsieh J Gauss G.H. Lohman T.M. Waksman G. Cell. 1997; 90: 635-647Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). 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 (10Subramanya H.S. Bird L.E. Brannigan J.A. Wigley D.B. Nature. 1966; 85: 379-383Google Scholar) 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 (12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). For example, poly(rU) enhancedkcat 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 (8Wong I. Moore K.J.M. Bjornson K.P. Hsieh J. Lohman T.M. Biochemistry. 1996; 35: 5726-5734Crossref PubMed Scopus (37) Google Scholar). Turnover numbers for HCV helicase and Rep helicase determined in the presence of single-stranded nucleic acid are similar (8Wong I. Moore K.J.M. Bjornson K.P. Hsieh J. Lohman T.M. Biochemistry. 1996; 35: 5726-5734Crossref PubMed Scopus (37) Google Scholar, 12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). 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 (12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), 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 (13Troullier A. Girardet J.-L. Dupont Y. J. Biol. Chem. 1992; 267: 22821-22829Abstract Full Text PDF PubMed Google Scholar, 14Antonny B. Chabre M. J. Biol. Chem. 1992; 267: 6710-6718Abstract Full Text PDF PubMed Google Scholar, 15Chabre M. Trends Biochem. Sci. 1990; 15: 6-10Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 16Werber M.M. Peyser Y.M. Muhlrad A. Biochemistry. 1992; 31: 7190-7197Crossref PubMed Scopus (125) Google Scholar). However, some phosphotransferases effectively bind nucleotide diphosphate in the presence of F− and Mg2+ without Al3+ or Be2+ as a cofactor (17Murphy A.J. Coll R.J. J. Biol. Chem. 1992; 267: 5229-5235Abstract Full Text PDF PubMed Google Scholar, 18Narayanan N. Su N. Bedard P. Biochim. Biophys. Acta. 1991; 1070: 83-91Crossref PubMed Scopus (31) Google Scholar, 19Murphy A.J. Hoover J.C. J. Biol. Chem. 1992; 267: 16995-17000Abstract Full Text PDF PubMed Google Scholar, 20Coll R.J. Murphy A.J. J. Biol. Chem. 1992; 267: 21584-21587Abstract Full Text PDF PubMed Google Scholar, 21Daiho T. Kubota T. Kanazawa T. Biochemistry. 1993; 32: 10021-10026Crossref PubMed Scopus (23) Google Scholar, 22Kubota T. Daiho T. Kanazawa T Biochim. Biophys. Acta. 1993; 1163: 131-143Crossref PubMed Scopus (25) Google Scholar, 23Antonny B. Bigay J. Chabre M. FEBS Lett. 1990; 268: 277-280Crossref PubMed Scopus (30) Google Scholar). 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. 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 ε258 = 14.4 mm−1cm−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 ε260 = 9.35 mm−1 cm−1 and ε258= 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 ε260 = 147 mm−1cm−1 and 379 mm−1cm−1, respectively. NaF and AlF3 were of the highest purity from Aldrich. HCV helicase was purified as described previously (12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The concentration of HCV helicase monomer was calculated with an ε280 = 88.5 mm−1 cm−1 (12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), which is based on (rU)15 binding sites. The standard buffer was 0.05m MOPS, 3.5 mm MgCl2 at pH 7.0. 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 (12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). 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 (24Boyer P.D. The Enzymes. 6. Academic Press, New York1962: 95-113Google Scholar) was not significant under these assay conditions. 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 (25Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar)). The bound nucleotide was quantitated by liquid scintillation counting using ICN EcoLume scintillation mixture. The time course for hydrolysis of ATP to ADP (P(t)) was described by Equation 1, whereVi 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 toVf. P(t)=Vi−Vfk(1−e−kobst)+VftEquation 1 The dependence on NaF concentration of the uncorrectedkobs 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. kobs=axEquation 2 kobs=axnEquation 3 The effect of pyruvate kinase concentration onkobs 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. kobs=a[pyruvate kinase]+bEquation 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, whereK was the dissociation constant of E for ADP. E+ADP⇌KE·ADPREACTION 1 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 ΔA∞ was the fractional activity decrease resulting from conversion of E toE·ADP. A([ADP])=1−ΔA∞[E·ADP][Et]Equation 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 wasn[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 Equation6. [E·ADP]=[ADP]+[nE]+K2−12([ADP]+[nE]+K)2−4[nE][ADP]Equation 6 If more than one binding site for ADP was present on the enzyme, the concentration of E was multiplied by n, wheren was a fitted parameter. This assumed that the ADP bound to each site on E independently. If the fit of Equations 5 and6 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)=Af+(1−Af)e−ktEquation 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), whereA([L]) was the ATPase activity at a ligand concentration of [L] (L was NaF or MgCl2), and ΔA∞ was the maximal decrease in activity at an infinite concentration of L. A([L])=1−ΔA∞[L]nKn+[L]nEquation 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([(dU)18])=1−1−ba[(dU)18],[(dU)18] 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 mmNaF, 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 ( 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 (t½ ∼ 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 μmsolution 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 ΔA∞ = 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). 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 ΔAf = 0.110 ± 0.008 and k = 0.0179 ± 0.0005 min−1. This corresponded to at½ for inhibition of 40 min. For comparison, inhibition of the ATPase activity under these conditions in the presence of excess poly(rU) had a t½ that was 500 s). In contrast to transducin, sarcoplasmic reticulum CaATPase and the Na,K-ATPase are potently inhibited by MgFxx −2complexes (17, 19 - 22). MgFxx −2 complexes were slowly time-dependent inhibitors that formed an inhibited enzyme species that reactivated slowly upon dilution (t½ = 6 min (1 mm Ca2+) and t½ = 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 (23Antonny B. Bigay J. Chabre M. FEBS Lett. 1990; 268: 277-280Crossref PubMed Scopus (30) Google Scholar), 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 (11Yao N. Hesson T. Cable M. Hong Z. Kwong A.D. Le H.V. Weber P.C. Nat. Struct. Biol. 1997; 4: 463-467Crossref PubMed Scopus (420) Google Scholar). In these complexes, MgFxx −2 is proposed to bind at the γ-phosphate binding site for the nucleotide triphosphate (23Antonny B. Bigay J. Chabre M. FEBS Lett. 1990; 268: 277-280Crossref PubMed Scopus (30) Google Scholar). 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 (12Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). 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 (t½ = 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% versus67%). 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 −2trapped 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 (27Berdis A.J. Benkovic S.J. Biochemistry. 1997; 36: 2733-2743Crossref PubMed Scopus (25) Google Scholar) 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 (ε280 = 88 mm−1cm−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 (28Stole E. Bryant F.R. Biochemistry. 1997; 36: 3483-3490Crossref PubMed Scopus (17) Google Scholar). Wong and Lohman (6Wong I. Lohman T.M. Biochemistry. 1997; 36: 3115-3125Crossref PubMed Scopus (25) Google Scholar) 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.