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J. Biol. Chem., Vol. 277, Issue 46, 43778-43784, November 15, 2002

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Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA^*

Yong-Joo Jeong, Dong-Eun Kim, and Smita S. Patel

From the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received for publication, August 22, 2002, and in revised form, September 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [¹⁸O]P_i exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P_i-release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P_i-release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase (dTTPase) reaction is the release of dTDP. Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer. The results of this study are therefore consistent with those reported earlier (Hingorani, M. M., Washington, M. T., Moore, K. C., and Patel, S. S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5012-5017), and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F₁-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Helicases are motor proteins that translocate along nucleic acids using the energy of NTP¹ hydrolysis. This activity facilitates many nucleic acid metabolic processes including those that require the separation of double-stranded DNA into their component single strands (1-3). A class of helicases assembles into ring-shaped hexamers, and helicases belonging to this class have been found to function in genome replication and recombination that require processive action of the helicase (1). The assembly of helicase subunits into hexamers is generally induced by NTP binding, and the hexamer is also stabilized by DNA (1, 4-6). Bacteriophage T7 gp4 protein is one of the better-studied ring helicases whose function is to facilitate T7 genome replication and recombination. T7 gp4 proteins gp4A' and gp4B assemble into rings in the presence of nucleotide triphosphate, preferably dTTP or its non-hydrolyzable analog dTMP-PCP. The hexamer has a high affinity for ssDNA that binds within the central channel of the ring. It has been proposed that the helicase passes only one strand of the double-stranded DNA through the central channel and excludes the complementary strand from the central channel as it translocates and separates the strands of the double-stranded DNA (7-10).

The translocation and strand separation activity of T7 helicase is fueled by dTTP hydrolysis. The mechanism by which dTTP hydrolysis is coupled to translocation and strand separation is not known. Our previous studies indicate that the dTTPase mechanism of gp4A' shows striking similarity to the binding change mechanism of the F₁-ATPase protein (11) despite there being no amino acid sequence homology between them. These studies showed that two-three dTTPs bound tightly but did not get hydrolyzed at the steady state rate, and these were referred to as the noncatalytic sites. Two additional dTTPs, referred to as catalytic sites, were hydrolyzed in a sequential manner. Based on these results, it was proposed that there are three noncatalytic and three catalytic sites on T7 helicase hexamer, similar to F₁-ATPase. The exact role of the noncatalytic sites has not been established, and it could be structural or regulatory. Since that report, pre-steady state kinetic studies of the ATPase reaction has been carried out with the hexameric Escherichia coli DnaB protein also in the absence of DNA, and the authors observed that three-four ATPs were hydrolyzed simultaneously (12). The authors proposed that all six sites of DnaB hydrolyze ATP simultaneously, and the observation of three-four-site hydrolysis was due to an unfavorable equilibrium constant for ATP hydrolysis on the active site of DnaB. The authors also raised several questions casting doubts on the studies with T7 helicase regarding hydrolysis of dTTP in a sequential manner. They questioned whether the observed kinetics were influenced by hexamer assembly. In addition, the question was raised of whether reversible dTTP hydrolysis reaction on the enzyme active site was responsible for the observed hydrolysis of one dTTP at a time.

We address several of these questions in this paper. We have now figured out conditions where we can preassemble the gp4A' hexamer. Hexamer assembly requires the presence of dTTP, but we have found that Mg²⁺ is not necessary for dTTP binding, hexamer formation, or DNA binding (6). Thus, stable gp4A' hexamers can be assembled in the presence of dTTP without Mg²⁺, conditions under which dTTP hydrolysis is very slow. Thus, the dTTPase kinetics can be studied with a preformed hexamer, where oligomerization will not limit the observed kinetics. Using a preassembled hexamer of T7 helicase, we have reexamined the mechanism of dTTP hydrolysis in the absence of DNA using pre-steady state kinetic methods. In addition, we have carried out a more detailed study of the dTTPase mechanism using medium [¹⁸O]P_i exchange experiments to explore the reversibility of the dTTPase reaction on the enzyme active site and have measured the rate of P_i release using a phosphate sensor protein. The findings of this study are consistent with those reported previously (11) and show that dTTP hydrolysis in the absence of DNA is sequential and, in addition, show that dTTP hydrolysis is not reversible on the enzyme active site. These studies support the previously proposed mechanism of dTTP hydrolysis by T7 helicase and also lay the basis for future studies in the presence of DNA.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

T7 Helicase-Primase gp4A' Protein, Nucleotides, and Buffer-- The T7 helicase used in this study is gp4A', which is a M64L mutant of T7 helicase-primase protein that was overexpressed and purified as described previously (13, 14). The protein concentration was determined both by absorbance measurements at 280 nm in 8 M urea (the extinction coefficient is 76,100 M¹ cm¹) and by the Bradford assay using bovine serum albumin as a standard. Both methods provided similar protein concentrations. dTTP and dTDP were purchased from Sigma, and [-³²P]dTTP was obtained from Amersham Biosciences. Tris buffer was used throughout the experiments, unless specified otherwise, which contained 50 mM Tris/Cl (pH 7.6), 40 mM NaCl, and 10%(v/v) glycerol.

Purification of MDCC-labeled Phosphate Binding Protein-- The single cysteine mutant (A197C) phosphate-binding protein (PBP) was expressed and purified as described (15, 16). PBP concentration was determined by absorbance measurement at 280 nm using an extinction coefficient of ₂₈₀ = 60,880 M¹cm¹. Labeling of the A197C PBP protein with MDCC and further purification of the labeled protein (PBP-MDCC) were performed as described (15) except a phosphate mop composed of 200 µM 7-methyl guanosine and 0.2 units/ml purine nucleoside phosphorylase was included in the labeling reaction. The labeled protein after purification showed a 280/430-nm absorbance ratio of 1.6, suggesting that most of PBP was labeled with MDCC (15). Molecular weight of the labeled PBP was measured by electrospray mass spectrometry (CUNY-Hunter College, New York, NY), and the correct molecular weight of PBP-MDCC (M_r = 34,847) was confirmed.

Phosphate-Water Oxygen Exchange Experiment-- [¹⁸O]P_i was prepared by reacting PCl₅ (from Sigma) with 99% H₂¹⁸O (Aldrich) followed by ion exchange chromatography on a Bio-Rad AG1-X4 column (17). The resulting phosphate had an enrichment of 96% ¹⁸O as determined by ³¹P NMR. The concentration of [¹⁸O]P_i was determined by the molybdate method (18).

Gp4A' (1 µM hexamer) was incubated with 10 mM MgCl₂, 0.5 mM EDTA, 6 mM dTDP, 20 mM [¹⁸O]P_i, and 60% (v/v) D₂O in 1 ml of 20 mM Tris buffer (pH 7.6) containing 50 mM NaCl and 10% glycerol at room temperature for 12 h. The reaction was quenched by vortexing with chloroform, and the aqueous phase was extracted. The distribution of P¹⁸O_i¹⁶O_4j species (0  j  4) was determined by ³¹P NMR (19). The spectra were obtained on a JEOL GX-400 instrument at a frequency of 162 MHz for phosphorus, and 500 scans were accumulated and Fourier-transformed. The fractional contribution of each [¹⁸O]P_i species to the spectrum was evaluated directly from the peak area. The observed ¹⁸O distribution in the phosphate were compared with those obtained from the control experiment, which was performed without gp4A'.

Pre-steady State Acid-quenched Kinetics of dTTP Hydrolysis-- The kinetic experiments were conducted on a rapid chemical quench-flow instrument at 18 °C (KinTek RQF3 software, State College, PA). First, gp4A' (4 µM hexamer) in Tris buffer was mixed with an equal volume of [-³²P]dTTP plus dTTP (240-1240 µM) and EDTA (10 mM). The mixture was immediately loaded into the quench-flow instrument, and in exactly 3 min, 24 µl was mixed with an equal volume of MgCl₂ (9.12-9.62 mM in the same buffer). After various times, the reactions were quenched with 4 M formic acid. An aliquot of the quenched reactions was spotted on polyethyleneimine-cellulose TLC and developed in 0.4 M potassium phosphate (pH 3.4) solution. Unreacted dTTP and product dTDP were quantitated using a PhosphorImager and ImageQuant (Molecular Dynamics). The molar concentration dTDP was plotted versus the time of reaction, and the data were fit to the burst equation (Equation 1),

(Eq. 1)

where D(t) is [dTDP] at time t, A is the amplitude of the burst phase, k₁ is the observed rate constant of the burst phase, and m is slope of the linear phase (steady state rate).

Pre-steady State Chase-time Kinetics of dTTP Hydrolysis-- The experiment was conducted on a rapid quench-flow instrument at 18 °C. Gp4A' (4 µM hexamer) in Tris buffer was mixed with an equal volume of [-³²P]dTTP plus dTTP (840 µM) and EDTA (10 mM). The mixture was loaded into the quench-flow instrument, and in 3 min, 24 µl of the gp4A' solution was mixed with an equal volume of dTTP chase (5.0 mM) and MgCl₂ (14.42 mM) in the same buffer. After various times, the reactions were quenched with 4 M formic acid, and the products were analyzed as above. The dTTP hydrolysis kinetic data were corrected for inefficient chase by subtracting the slow linear increase after the exponential phase. The resulting chase kinetics was fit to the sum of two exponentials (Equation 2),

(Eq. 2)

where D(t) is [dTDP] at time t, A1 and A2 are the amplitudes of each exponential phase, and k₁ and k₂ are the observed rate constants of each exponential phase.

Pre-steady State Kinetics of Inorganic Phosphate Release-- Fluorescence stopped-flow kinetic experiments were performed using an instrument manufactured by KinTek Corp. (State College, PA). Inorganic phosphate release reactions were performed in Tris buffer at 18 °C. Because PBP-MDCC is sensitive to nanomolar quantities of P_i ubiquitously contaminated in all solutions, a coupled enzyme reaction (phosphate mop: 0.5 units/ml purine nucleoside phosphorylase and 300 µM 7-methyl guanosine) was used to sequester P_i chemically as ribose-1-phosphate. Assay conditions were adjusted to ensure that the phosphate mop did not compete with PBP-MDCC for phosphate (k_cat/K_m for the purine nucleoside phosphorylase reaction with P_i is 3.2 × 10⁶ M¹s¹) (20). The fluorescence signal of PBP-MDCC was calibrated using P_i standards on the stopped-flow apparatus. The amplitude of fluorescence increase was measured by conducting a control experiment in the absence of P_i and subtracting the maximum fluorescence of the control from one with a known concentration of P_i. The amplitude thus calculated was plotted versus P_i concentration to create the calibration curve. The calibration curve was created using the same photomultiplier tube voltage as used in the subsequent experiment just before a set of P_i-release kinetics were measured. The slope was used to convert the observed fluorescence amplitude into molar P_i.

A 40-µl solution containing gp4A' (0.4 µM hexamer), EDTA (5 mM), P_i mop (0.5 units/ml purine nucleoside phosphorylase with 300 µM 7-methyl guanosine), and dTTP (100-400 µM) was rapidly mixed with 40 µl of MgCl₂ (4 mM), P_i mop, and 10 µM PBP-MDCC in the stopped-flow instrument at 18 °C. The fluorescence changes of PBP-MDCC were monitored using an excitation wavelength of 425 nm and monitoring the emission above 450 nm using a cut-off filter (Corion LL-450 F). For each experiment, at least four fluorescence traces were averaged. The fluorescence changes were converted to the concentration of released phosphate using the standard curve. The concentration of released phosphate per gp4A' hexamer was plotted versus time of reaction, and the data were fit to the burst equation (Equation 1).

Steady State Chase-time Kinetics of dTTP Hydrolysis-- The experiment was conducted at 30 °C using two delay times on a rapid quench-flow instrument. Gp4A' (4 µM hexamer) in Tris buffer was mixed with an equal volume of [-³²P]dTTP (400 µM) and EDTA (10 mM). The mixture was loaded into the quench-flow instrument, and in 3 min, 24 µl of the gp4A' solution was mixed with an equal volume of MgCl₂ (9.2 mM) in the same buffer for 15 s. After 15 s, a solution containing unlabeled dTTP (20 mM) and MgCl₂ (22 mM) in the same buffer was added from the third syringe, and the time was varied before the reactions were quenched with 4 M formic acid. The quenched reactions were analyzed by TLC and dTTP hydrolysis due to inefficient chase that exhibits a slow linear increase after the exponential phase was subtracted. The resulting chase kinetic data were fit to Equation 3,

(Eq. 3)

where D(t) is [dTDP] at time t, A is the amplitude, and k₁ is the observed rate constant.

Global Fitting of dTTPase and P_i-release Kinetics-- The pre-steady state acid-quenched kinetics of dTTP hydrolysis and P_i-release kinetics at various [dTTP] were globally fit to the kinetic model shown in Table II using the software Scientist (MicroMath Research, Salt Lake City, UT). The concentration of each species (T, dTTP; D, dTDP; P, P_i; H, T7 hexamer; HT, T7 hexamer-dTTP complex; HDP, T7 helicase-dTDP·P_i complex; and HD, T7 helicase-dTDP complex) as a function of time was determined by numerical integration of the differential equations describing the mechanism. The determined rate constants (k₂/k₁, k₄/k₃, k₅, k₆, k₇, and k₈) are the best-fit parameters, obtained by globally fitting the combined data simultaneously.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The minimal pathway of helicase-catalyzed dTTP hydrolysis consists of the following steps; 1) dTTP binding to T7 helicase hexamer, 2) dTTP hydrolysis to dTDP and P_i, 3) release of P_i, and 4) release of dTDP. The aim of this paper is to determine the rate of dTTP hydrolysis, its equilibrium constant on the enzyme, the rate of P_i release, and the rate of dTDP release from T7 helicase in the absence of ssDNA. The measurement of these kinetic parameters allows us to elucidate the complete kinetic mechanism of dTTP hydrolysis by the hexamer in the absence of DNA, to determine the rate-limiting step in the pathway of dTTP hydrolysis, and to determine whether there is any cooperativity in dTTP hydrolysis by the subunits of the hexamer.

Pre-steady State Kinetics of dTTP Hydrolysis-- Previous studies show that gp4A' assembles into a hexamer in the presence of dTTP without Mg²⁺, and under these conditions dTTP hydrolysis occurs at a very slow rate of 1.7 × 10⁴ s¹ (6). The hexamer was assembled by incubating gp4A' with radiolabeled dTTP in the absence of Mg²⁺ (6). After exactly 3 min, the dTTPase reaction was initiated by rapidly mixing the hexamer with MgCl₂ in a quench-flow instrument, and the reactions were acid-quenched after millisecond to second times (Fig. 1A). The 3-min preincubation period was chosen because it is the shortest time within which we can mix and load the syringes of the quench-flow instrument. We determined by experimentation that 3-min incubation of gp4A' with dTTP was sufficient for dTTP binding and hexamer formation (data not shown). A control experiment was performed, and the amount of dTTP hydrolyzed in the preincubation time period was subtracted from the final data.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Pre-steady state acid-quenched kinetics of dTTP hydrolysis. A, the schematic shows the design of the acid-quenched experiment. Equal volumes of gp4A' (4 µM hexamer) and a mixture of dTTP and [-32P]dTTP and EDTA (10 mM) were incubated for 3 min. The preformed hexamer was then mixed with an equal volume of MgCl2 (9.12-9.62 mM) to initiate dTTP hydrolysis, and after various times, the reactions were acid-quenched. The final concentrations of gp4A' and free MgCl2 were 1 µM hexamer and 2 mM, respectively. B, shows a representative kinetic trace of dTTP hydrolysis with 160 µM dTTP. The dTTP hydrolysis kinetics fit best to the burst equation (Equation 1), as shown by the continuous line. The pre-steady state dTTP hydrolysis experiments were repeated at different concentrations of dTTP (final concentration of 60, 110, 210, and 310 µM). The [dTTP] dependence of the observed burst rate (C) and burst amplitude (D) are shown. The burst rate versus [dTTP] fit to a hyperbola with a maximum dTTP hydrolysis rate of 23.6 ± 4.1 s1 and K1/2 of 135.0 ± 54.4 µM.

As shown in Fig. 1B, the acid-quenched pre-steady state kinetics of dTTP hydrolysis is biphasic, showing a burst of dTTP hydrolysis followed by a linear increase at the steady state rate. The burst kinetics indicate that hydrolysis of dTTP on the active site of the helicase is faster than the subsequent steps of product release or dTTP rebinding. Previous studies show that dTTP binding occurs at a rate >7 × 10⁵ M¹s¹ (11), and thus, under the conditions of the experiments ([dTTP] ranging from 60 to 310 µM), the rate-limiting step would be the product release step/s. To estimate the rate of dTTP hydrolysis, the acid-quenched kinetics were measured at increasing [dTTP] (final, 60-310 µM). The observed burst rate constant increased in a hyperbolic manner with increasing [dTTP] providing a saturating rate of dTTP hydrolysis (23.6 ± 4.1 s¹) and dTTP K_1/2 (135.0 ± 54.4 µM) (Fig. 1C). At 310 µM dTTP, well above the K_m of dTTPase (10 µM), the observed pre-steady state burst amplitude was close to 0.8 sites per hexamer (Fig. 1D). Thus the acid-quenched pre-steady state dTTPase kinetics indicates that dTTP hydrolysis on the helicase active site is faster than the subsequent steps of product release and the burst amplitude being close to one indicates that only one dTTP is hydrolyzed during the first turnover.

¹⁸O Exchange between Inorganic Phosphate and Water-- We have used the [¹⁸O]P_i medium exchange experiments to determine whether dTTP hydrolysis on the hexamer active site is reversible. The [¹⁸O]P_i exchange method, developed by Boyer and Hutton (21), has been used to analyze the reversibility of the hydrolysis step of many ATPases. Briefly, high concentrations of dTDP (6 mM) and [¹⁸O]P_i (20 mM) were mixed with gp4A', and incorporation of unlabeled oxygen from water into P_i due to any reversible dTTPase reaction was monitored with time. The loss of ¹⁸O label in P_i was monitored by ³¹P NMR experiments. It is already known that ¹⁸O bound to the phosphorous atom shifts the ³¹P NMR by about 0.02 ppm up-field compared that of the ¹⁶O species (19). A control experiment was conducted without gp4A' to obtain the distribution of [¹⁸O]P_i species in the absence of enzyme. As shown in Table I, even after 12 h of incubation with T7 helicase, the P¹⁸O_j¹⁶O_4j species distribution (where 0  j  4) did not change, indicating the absence of dTTP synthesis on the helicase active site and showing irreversibility of the dTTP hydrolysis step. The absence of medium [¹⁸O]P_i exchange may be due to the weak binding of P_i to the gp4A'·dTDP species.

Pre-steady State Kinetics of P_i Release-- The burst kinetics of dTTP hydrolysis in the acid-quenched experiment, discussed above, indicated that one or both product release steps are rate-limiting. To determine the rate of P_i release, we have used the real time P_i-release stopped-flow assay developed by Webb and coworkers (15). The P_i sensor is the phosphate-binding protein labeled with MDCC (PBP-MDCC). The binding of P_i to PBP occurs at a rapid rate (1.4 × 10⁸ M¹s¹) and with a high affinity (K_d = 0.1 µM) and results in an increase in PBP-MDCC fluorescence. Thus, the rate of P_i release can be measured in real time in a stopped-flow instrument by following the increase in fluorescence of MDCC (15).

As shown in Fig. 2A, gp4A' was preincubated with various concentrations of dTTP (in the absence of Mg²⁺) and mixed with MgCl₂ and PBP-MDCC in a stopped-flow instrument at 18 °C. The magnitude of fluorescence increase was converted to P_i concentration, which was plotted as a function of time (Fig. 2B). A pre-steady state burst of P_i release was observed at all concentrations of dTTP, which indicated that the P_i-release rate is faster than the dTTPase turnover rate, which must be limited by the dTDP release rate. The burst amplitude of P_i release is close to one (Fig. 2C) and is the same as the burst amplitude observed in the acid-quenched experiments, indicating that only one of six hexameric sites hydrolyze dTTP at a time. The P_i-release rate at saturating [dTTP] is 2.7 s¹ (Fig. 2D), which is about 10-fold slower than the observed dTTP hydrolysis rate (24 s¹). This is more clearly seen in Fig. 3, where we compare the kinetic traces of dTTP hydrolysis and the P_i release under the same conditions. Because the P_i-release rate is only slightly faster than the steady state rate of dTTP hydrolysis (1.8 s¹), we conclude that both P_i-release and dTDP-release steps are slow, but dTDP release dictates the observed dTTPase turnover rate without DNA.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Pre-steady state kinetics of Pi-release. A, the schematic shows the design of the stopped-flow Pi-release experiment. Gp4A' (0.4 µM hexamer), EDTA (5.0 mM), and dTTP (100-400 µM) was mixed with MgCl2 (9.1-9.4 mM) and PBP-MDCC (10 µM) in a stopped-flow instrument at 18 °C. The final concentrations of gp4A' and free MgCl2 were 0.2 µM hexamer and 2 mM, respectively. The fluorescence (em > 450 nm) upon excitation at 425 nm was measured as a function of time. B, the representative kinetic trace shows the molar amount of Pi released per mole of gp4A' hexamer for an experiment performed with 100 µM dTTP. The kinetics fit best to the burst equation (Equation 1). The [dTTP] dependence of the burst amplitude (C) and rate constants (D) is shown. The burst amplitudes remained constant at an average value of 0.93 ± 0.05 sites per hexamer (dashed line), and the burst rate around 2.7 s1.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Pre-steady state kinetics of dTTP hydrolysis and Pi-release; global analyses. Representative kinetic traces from the acid-quenched (filled circle) and Pi-release experiments are shown. The dashed lines are simulated curves obtained using the best-fit rate constants shown in Table II.

Pre-steady State Chase-time Kinetics of dTTP Hydrolysis; Two dTTPs Are Hydrolyzed Sequentially-- Both the acid-quenched pre-steady state experiment and the P_i-release kinetics indicate that T7 helicase hexamer on an average hydrolyzes only one dTTP in a single turnover. To determine whether additional sites hydrolyze dTTP in subsequent turnovers, pre-steady state chase experiments were designed. The chase experiment can potentially reveal how many dTTPs are tightly bound to the hexamer before hydrolysis, their hydrolysis rates, and whether they are hydrolyzed sequentially or simultaneously. The experiment was carried out as shown in Fig. 4A by mixing gp4A' with [-³²P]dTTP in the absence of MgCl₂ for 3 min, during which time dTTP binds and the hexamer is assembled. The hexamer bound to radiolabeled dTTP was then mixed in a quench-flow instrument with an equal volume of a high concentration of non-radiolabeled Mg-dTTP that acts as a chase. After various times of mixing with the chase, the reactions were acid-quenched. The observed biphasic kinetics of dTTP hydrolysis during the chase-time is shown in Fig. 4B. The biphasic chase kinetics best fit to the sum of two exponentials, which indicated that close to 1 dTTP (0.85 ± 0.07 dTTP/hexamer) hydrolyzes with a rate constant of 18.0 ± 3.5 s¹, and a second dTTP (0.67 ± 0.05 dTTP/hexamer) hydrolyzes with a rate constant of 0.58 ± 0.1 s¹. Note that the fast rate of dTTP hydrolysis in the chase-time experiment is close to the rate of dTTP hydrolysis in the acid-quenched experiment (Fig. 1B), whereas the slow rate of dTTP hydrolysis is close to the steady state rate (1.8 s¹). Consistent with the previously reported mechanism of dTTPase in the absence of ssDNA (11), the chase-time kinetics indicates that at least two dTTPs are hydrolyzed sequentially by T7 helicase hexamer. One dTTP is hydrolyzed at the intrinsic hydrolysis rate, whereas the hydrolysis of the second dTTP is limited by the rate of product release from the first site.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Pre-steady state chase-time kinetics of dTTP hydrolysis. A, Gp4A' (4 µM hexamer) was incubated with [-32P]dTTP (840 µM) in the presence of EDTA (10 mM) for 3 min, and then the solution was rapidly mixed with MgCl2 (14.42 mM) and unlabeled dTTP (5 mM) to start the reaction. The final concentrations of gp4A', [-32P]dTTP, and free MgCl2 were 1 µM hexamer, 210 µM, and 2 mM, respectively. B, shows the hydrolysis of dTTP as a function of chase time that fits best to the sum of two-exponentials (Equation 2). The fast phase has an amplitude of 0.85 ± 0.07 dTTP per hexamer with an exponential rate constant of 18.0 ± 3.5 s1; the slower phase has an amplitude of 0.67 ± 0.05 dTTP per hexamer with an exponential rate constant of 0.58 ± 0.1 s1. The inset shows dTTP hydrolysis in the fast phase, and the dashed line shows the poor fit of the entire kinetic trace to one-exponential.

Steady State Chase-time Kinetics of dTTP Hydrolysis; two dTTPs Are Hydrolyzed Simultaneously-- The above pre-steady state experiments indicate that of the six dTTP binding sites on the hexamer, at least two sites hydrolyze dTTP in a sequential manner. Most hexameric helicases show two classes of NTP binding sites (1). It was observed that half of the sites bind NTP tightly, and the other half bind NTP weakly due to negative cooperativity in nucleotide binding. We have proposed that three sites of T7 helicase hexamer are catalytic, and they hydrolyze dTTP sequentially. There are two to three additional sites on the hexamer that we proposed are "noncatalytic"; that is, they bind dTTP but do not hydrolyze them at a significant rate. Here we have designed a chase experiment to obtain further evidence for the noncatalytic sites and to determine the rate at which they hydrolyze bound dTTP. As shown in Fig. 5A, gp4A' was mixed with [-³²P]dTTP for 3 min to preform the hexamer and then mixed with MgCl₂ for 15 s in the quench-flow instrument to allow enzyme catalysis to reach steady state. After exactly 15 s, when the dTTPase reaction with radiolabeled dTTP was in steady state, an excess of non-radioactive dTTP was added, and after varying chase times, the reactions were acid-quenched. We carried out this experiment at 30 °C to accelerate the rate so the chase-time kinetics could be measured within 65 s because our instrument does not allow us to vary chase times greater than 65 s.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Steady state chase-time kinetics of dTTP hydrolysis. A, Gp4A' (4 µM hexamer), [-32P]dTTP (400 µM), and EDTA (10 mM) were incubated for 3 min, loaded in the quench-flow instrument, and then rapidly mixed with MgCl2 (9.2 mM) at 30 °C for 15 s to allow the dTTPase reaction to reach steady state. The final concentrations of gp4A', [-32P]dTTP, and free MgCl2 were 1 µM hexamer, 100 µM, and 2 mM, respectively. After 15 s, non-radiolabeled dTTP chase (20 mM) and MgCl2 (22 mM) were added, and the reaction was acid-quenched after various chase times. B, the hydrolysis of dTTP during the chase-time was measured and corrected for inefficient chase. The resulting chase kinetics fit best to one-exponential (Equation 3) with an amplitude of 1.9 ± 0.3 dTTP per hexamer and an exponential rate of 0.11 ± 0.05 s1.

Fig. 5B shows a representative chase-time kinetic trace corrected for inefficient chase. Interestingly, simultaneous hydrolysis of two radiolabeled dTTPs was observed at a rate that is much slower than the dTTP hydrolysis rate observed in the chase-time kinetics under pre-steady state conditions (see Fig. 4B). The kinetics fit to an equation describing one exponential with an amplitude corresponding to close to two dTTPs (1.9 ± 0.3)/hexamer and an exponential rate constant of 0.11 ± 0.05 s¹. We estimate that the noncatalytic sites hydrolyze dTTP at a rate close to 500 times slower than the hydrolysis rate at the catalytic sites (by comparing the chase rate in Fig. 4 with the chase rate in Fig. 5, adjusted for temperature difference). The steady state dTTPase rate was measured at 30 °C as 3.5 s¹. Thus, dTTPs at the noncatalytic sites get hydrolyzed at a rate that is 32 times slower than the dTTPase turnover rate at the catalytic sites. This result is consistent with the previously reported results (11) and indicates that there are at least two noncatalytic or slowly hydrolyzing dTTPase sites that bind dTTP tightly but hydrolyze them at a slow rate relative to the hydrolysis and turnover rate of dTTP at the catalytic sites. The exact role of these slowly hydrolyzing sites on T7 helicase hexamer is not known, and we speculate that they may be structural (holding the hexamer structure) or regulatory sites.

Global Analysis of dTTPase and P_i-release Kinetics-- The acid-quenched (Fig. 1) and P_i-release kinetics (Fig. 2) at various [dTTP] were globally fit to the model shown in Table II. This model is the same as the one proposed earlier (11). As shown in Fig. 6, the model consists of the following steps; sequential binding of two dTTPs to the hexamer, hydrolysis of one dTTP followed by P_i release and dTDP release, and then the repetition of the cycle. The global fit provided the rate constants summarized in Table II with standard deviations. The dashed lines in Fig. 3 are the predicted or simulated lines for the particular dTTP concentration derived from the best-fit parameters obtained by global fitting. The dTTP synthesis step rate constant, k₄, was assumed to be zero based on the results of [¹⁸O]P_i-exchange experiments. The initial concentrations (at time 0) of free hexamer and dTTP-bound hexamer were floated during the global fitting, which reflects the fraction of dTTP-bound state of gp4A' hexamer in the absence of Mg²⁺ before initiation of the hydrolysis reaction. Global fitting provided a dTTP K_d (equal to k₂/k₁) of 10.0 ± 0.6 µM for one dTTP binding and a K'_d (equal to k₄/k₃) of 201.5 ± 33.1 µM for a second dTTP binding in sequence, and for the rate of dTTP hydrolysis, k₅ = 41.4 ± 2.0 s¹, P_i release, k₇ = 5.3 ± 0.05 s¹, and dTDP release, k₈ = 1.7 ± 0.01 s¹. The rates of dTDP and P_i rebinding to the hexamer could not be determined because under the conditions of the experiment (pre-steady state) these rates would be negligible.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Mechanism of dTTP hydrolysis by T7 helicase. The clear and shaded subunits represent catalytic and noncatalytic dTTPase sites, respectively, shown to be alternating, although the exact configuration cannot be determined from our experiments. T, dTTP; D, dTDP; D-P, dTDP·Pi. The sequential hydrolysis of two dTTPs at the catalytic sites is shown with intrinsic rate constants that were determined by globally fitting the kinetic data reported in this paper (Table II).

	DISCUSSION

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Previous pre-steady state kinetics of dTTP hydrolysis of T7 helicase gp4A' indicated that although T7 helicase binds three-four dTTPs per hexamer, only one dTTP is hydrolyzed at a time in the absence of DNA, and sequential hydrolysis of two dTTPs was observed in a chase experiment (11). From those results we inferred that T7 helicase hydrolyzed dTTP in a sequential manner in the absence of DNA. In this paper, we reinvestigate the kinetic pathway of dTTP hydrolysis using the assembled hexamer of gp4A'. T7 helicase requires the presence of dTTP to form a stable hexamer. We have recently shown that gp4A' assembles into stable hexamers in the presence of dTTP without Mg²⁺ (6). All the experiments were carried out by preincubating gp4A' and dTTP for 3 min in advance to make the starting oligomeric state as a pre-assembled hexamer. In addition, we have carried out [¹⁸O]P_i medium exchange experiments to determine whether dTTP hydrolysis is reversible on the enzyme active site and have characterized the rate-limiting steps by measuring the kinetics of P_i-release. The studies reported in this paper, therefore, provide a complete description of the kinetic pathway of dTTP hydrolysis by the hexameric gp4A' helicase and provide the basis to study the mechanism in the presence of DNA.

Consistent with previously reported studies (11), we find that even with a preassembled hexamer with dTTP bound, only one-sixth of the potential dTTP binding sites on the hexamer hydrolyze dTTP at a fast rate. This is evident from the magnitude of the burst phase of the acid-quenched, P_i-release, and chase kinetic experiments. The observed amplitude of one dTTP per hexamer is smaller than the six potential dTTP binding sites of the hexamer, and one reason this could be is if hydrolysis of dTTP on the helicase active site were reversible. Thus, if the rate of dTTP synthesis were much faster than the rate of dTTP hydrolysis on the helicase active site, then one would observe substoichiometric burst amplitude. To determine whether dTTP hydrolysis was reversible on the active site, we carried out [¹⁸O]P_i medium exchange experiments. These experiments indicated that the synthesis of dTTP on the active site is undetectable. This indicates that in the absence of ssDNA, the dTTP hydrolysis reaction on the helicase active site is almost irreversible. A second reason for not observing a stoichiometric burst amplitude is if part of the enzyme were inactive. Two types of active site titrations were routinely performed on all enzyme preparations, titration with dTTP and ssDNA. The enzyme from all preparations consistently showed 3-4 dTTPs tightly bound per hexamer and 1 ssDNA strand (25-30-mer) per hexamer. Thus, it does not appear that the enzyme preparations contain significant fractions of inactive enzyme, and the burst amplitude of close to one site per hexamer observed in the pre-steady state experiments reflects the actual number of active sites that hydrolyze dTTP hydrolysis in a single turnover.

The pre-steady state chase-time experiments also support the hypothesis that T7 helicase hexamer hydrolyzes one dTTP at a time; since these experiments also showed on an average that only one-sixth of the potential dTTP binding sites hydrolyze dTTP at a fast rate. In addition, the pre-steady state chase-time experiments showed that two dTTPs hydrolyze in a sequential manner. One dTTP is hydrolyzed at a fast rate, and the hydrolysis of the second dTTP occurs at a rate that is comparable with the steady state dTTPase rate. Thus, it appears that the release of product from one site triggers the hydrolysis of dTTP bound to another site. The product release step that triggers the conformational change in the hexamer that allows hydrolysis at another site could be either the P_i-release or the dTDP-release step. Using a P_i-sensor protein, PBP-MDCC, we have measured the real time kinetics of P_i-release, which indicated that P_i-release is about 3 times faster than the steady state rate. These results indicate that dTDP release is the rate-limiting step; it must cause a conformational change that is communicated between the subunits of the hexamer.

The steady state chase-time dTTPase kinetics indicated that in addition to dTTP hydrolysis occurring at the two catalytic sites there are two sites that hydrolyze dTTP at least 30 times slower than the steady state rate. The steady state chase-time experiment therefore provides additional evidence for noncatalytic or slowly hydrolyzing sites proposed previously for T7 helicase (11). Based on all the results thus far, we propose a detailed mechanism of dTTP hydrolysis by T7 helicase hexamer in the absence of DNA, which is shown in Fig. 6. This mechanism is very similar to the binding change mechanism of F₁-ATPase (22, 23). We propose that 3 sites on the hexamer are noncatalytic, and those bind dTTP tightly but hydrolyze them at a very slow rate during the steady state phase. These sites may serve to hold the hexamer together or to act as regulatory sites. The reason for observing hydrolysis of only two dTTPs might be that the third dTTP exchanges with medium nucleotide during the chase time. There is cooperativity in dTTP binding and hydrolysis at the catalytic sites. At a given time, one dTTP is bound tightly (with a K_d close to 10 µM), and a second one is loosely bound (K_d of ~200 µM). The binding of dTTP at the weaker site is necessary for hydrolysis of dTTP at the tight site (as evidenced from the observed K_1/2 of burst rate versus [dTTP] in the acid-quench experiments, which is close to the K_d of the second dTTP (Fig. 1C)). The dTTP is hydrolyzed at the tight site at a rate estimated to be close to 40 s¹ followed by P_i release at 5 s¹ and dTDP release at 1.7 s¹, which then triggers the tight binding and hydrolysis of dTTP at a second site. The cooperativity in binding, hydrolysis, and product release steps assures sequential hydrolysis of dTTP at two catalytic sites. We propose that sequential binding and hydrolysis of dTTP continues at the third site as well, although we do not have evidence for that and cannot directly measure the hydrolysis at the third site experimentally.

A recent crystal structure of T7 gp4 helicase domain (lacking the primase domain) in the absence of DNA shows that this domain assembles into a ring and binds nucleotide at the subunit interface (24). In the hexameric structure of the helicase domain, two different electron densities for bound ATP were observed. Two of the six NTP binding sites of the hexamer were found to bind NTP with a higher occupancy, two sites bound ATP with a lower occupancy, and the remaining two sites were empty. Based on this structure, a four-site binding change model was proposed. It was proposed that hydrolysis and binding of two NTPs take place simultaneously, and each pair of sites undergo out-of-phase NTP hydrolysis, nucleoside diphosphate plus P_i dissociation, and NTP binding steps. This model is not consistent with the previously reported kinetic data (11) or those in this paper, both of which show that on an average only one dTTP is hydrolyzed per hexamer and not two in the absence of DNA. Note that our studies are done with the full-length helicase-primase protein, and the kinetic mechanism of ATP hydrolysis by the helicase domain alone has not been characterized. Thus, it is possible that the two proteins hydrolyze dTTP with different kinetics and, very likely, that the dTTPase sites of the full-length protein are conformationally different from those of the truncated helicase domain protein.

One would eventually want to determine whether the NTPase mechanism is conserved among hexameric helicases. A survey of the nucleoside triphosphatase studies of hexameric helicases shows that the number of NTPs hydrolyzed in the pre-steady state burst phase is not consistent. For example, in the absence of DNA, T7 helicase hydrolyzes one dTTP per hexamer (this study and Ref. 11), E. coli DnaB helicase hydrolyzes three-four ATPs simultaneously (12), and RuvB helicase hydrolyzes two ATPs (25). In the presence of RNA, Rho protein has been shown to hydrolyze one to three ATPs per hexamer (26, 27). Similarly, sequential hydrolysis of NTP has been proposed for T7 helicase in the absence of DNA (11) and for Rho protein in the presence of RNA (27). The reason for the different number of nucleotides hydrolyzed in the first turnover by these hexameric helicases is not clear. It is possible that the pre-steady state phase is not representative of what happens in subsequent turnovers, and nucleotide hydrolysis in the first turnover represents an event unique to a hexameric helicase and involves steps related to hexamer assembly. Another reason is also that a detailed investigation of the nucleoside triphosphatase pathway has not been carried out for these helicases. For instance, although reversible hydrolysis of ATP was proposed to occur on the DnaB active site (12), experiments such as [¹⁸O]P_i exchange were not carried out to obtain direct evidence for reaction reversibility. Similarly, the pre-steady state experiments of DnaB were stopped by adding EDTA that acts more like a chase rather than a rapid quench, and this might be the reason why the burst amplitude was large. Clearly, more detailed studies are necessary to determine whether the nucleoside triphosphatase mechanism is conserved among hexameric helicases. Similarly, studies are necessary to understand the mechanism of NTP hydrolysis in the presence of nucleic acid. The NTPase pathway combined with the understanding of how the protein moves along nucleic acid will provide a better understanding of this remarkable molecular motor.

	ACKNOWLEDGEMENT

We thank Dr. Blumenstein for advice of ³¹P NMR studies (Department of Chemistry, Hunter College, New York, NY).

	FOOTNOTES

^* This research was supported by National Institutes of Health Grant GM55310 (to S. S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-3372; Fax: 732-235-4783; E-mail: patelss@umdnj.edu.

Published, JBC Papers in Press, September 10, 2002, DOI 10.1074/jbc.M208634200

	ABBREVIATIONS

The abbreviations used are: NTP, nucleoside triphosphate; dTMP-PCP, deoxythymidine (,-methylene)triphosphate; ssDNA, single-stranded DNA; P_i, inorganic phosphate; PBP, phosphate-binding protein; MDCC, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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