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J. Biol. Chem., Vol. 275, Issue 24, 17962-17967, June 16, 2000

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Equilibrium and Kinetic Studies of Substrate Binding to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Escherichia coli^*

Alun Bermingham, Joanna R. Bottomley^§, William U. Primrose^§, and Jeremy P. Derrick^¶

From the  Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, United Kingdom and ^§ PanTherix Ltd., Kelvin Campus, West of Scotland Science Park, Glasgow G20 0SP, United Kingdom

Received for publication, January 12, 2000, and in revised form, March 29, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue ,-methyleneadenosine triphosphate (AMPCPP) with high affinity. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) exhibited a substantial fluorescence enhancement upon binding to HPPK, with an equilibrium dissociation constant comparable with that for ATP (10.4 and 4.5 µM, respectively). The apoenzyme did not bind the second substrate HMDP, however, unless AMPCPP was present, suggesting that the enzyme binds ATP first, followed by HMDP. Equilibrium titration of HPPK into HMDP and AMPCPP showed an enhancement of fluorescence from the pterin ring of the substrate, and a dissociation constant of 36 nM was deduced for HMDP binding to the HPPK·AMPCPP binary complex. Stopped flow fluorimetry measurements showed that the rate constants for the binding of MANT-ATP and AMPCPP to HPPK were relatively slow (3.9 × 10⁵ and 1.05 × 10⁵ M¹ s¹, respectively) compared with the on rate for binding of HMDP to the HPPK·AMPCPP binary complex. The significance of these results with respect to the crystal structures of HPPK is discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacteria, fungi, and protozoa have the ability to synthesize folic acid de novo, whereas mammals must obtain it from their diet. For this reason, the enzymes of folic acid biosynthesis are an attractive group of potential antimicrobial drug targets. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK¹; EC 2.7.6.3) catalyzes a vital step in the folate biosynthesis pathway, the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP; see Fig. 1). DHPPP is a substrate for the next enzyme in the pathway, dihydropteroate synthase (DHPS; EC 2.5.1.15), which catalyzes the condensation of DHPPP with para-aminobenzoic acid to form dihydropteroate and pyrophosphate. Work by Brown and co-workers established that in Escherichia coli HPPK and DHPS are two separate enzymes that can be separated chromatographically (1-3). More recently, the purification of both enzymes to homogeneity from E. coli has been described (4), and subsequently the gene for HPPK was cloned and sequenced (5). Cloning and expression studies on the sulD gene from Streptococcus pneumoniae have shown that this organism encodes a bifunctional enzyme, with HPPK and dihydroneopterin aldolase activities (6, 7). (Dihydroneopterin aldolase catalyzes the previous step in the pathway to form the HPPK substrate HMDP.) Expression and partial purification of the S. pneumoniae enzyme showed that it is much larger than E. coli HPPK, being a trimer or tetramer of subunit molecular weight 31,000. The enzyme has, however, a relatively high degree of sequence conservation, and homologues have been identified from a variety of organisms (8-16).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Reaction catalyzed by HPPK.

The crystal structures of HPPK from Haemeophilus influenzae and E. coli have been reported to resolutions of 2.1 and 1.5 Å, respectively (14, 15). The enzymes from both organisms adopt an / structure, with five helices packed around a four-strand antiparallel -sheet. The fold generates a large crevice on the enzyme surface, approximately 26 Å long and 10 Å wide, which has been identified as the active site of the enzyme. A substrate analogue of HMDP was used to identify the principal residues involved in the recognition of the pterin ring; all of the potential hydrogen bond donors and acceptors in the pterin moiety are involved in hydrogen bonds to main chain or side chain atoms within the protein (14). The crystal structure of a nonproductive ternary complex has also been reported of E. coli HPPK in complex with ATP and an analogue of HMDP (16). The side chains from two Asp residues are involved in chelating two magnesium ions that lie adjacent to the -, -, and -phosphates within ATP and may play a role in stabilizing the reaction transition state.

Although the crystal structures have provided valuable information about the tertiary structure of HPPK, more detailed enzymological information concerning ligand binding affinities for the enzyme is scant. Previous investigators have reported K_m values for the two substrates, but the substrate binding order for HPPK has not been investigated (4, 13, 18). Here we show how the binding of both substrates to HPPK can be probed using fluorimetric measurements, allowing the measurement of equilibrium binding constants and some pre-steady state kinetic rate constants. The results are consistent with a compulsory order ternary complex mechanism, with ATP binding first in a relatively slow step, followed by the faster addition of HMDP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- The following chemicals were obtained from Sigma: ATP, AMP, GTP, AMPCPP, GMP, Hepes, inorganic pyrophosphatase, and purine nucleoside phosphorylase. 6-Hydroxymethylpterin was from Schircks Laboratories (Jona, Switzerland). 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) was from Molecular Probes, Inc. (Eugene, OR). 2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG) was prepared from ()-2-amino-6-chloropurine riboside (Aldrich), methyl iodide, and thiourea by the method of Webb (19). 6-Hydroxymethylpterin was reduced to HMDP by an adaptation of the method of Futterman (20).

Overexpression and Purification of E. coli HPPK-- The gene for HPPK was amplified by polymerase chain reaction from E. coli genomic DNA using the following two primers: 5'-GGCAGCGAATTCATATGACAGTGGCGTATATTGCCATAG-3', coding for the N terminus of the protein, with an NdeI site underlined, and 5'-GCAGCGGGATCCAAGCTTTTACCATTTGTTTAATTTGTCAAATGCTC-3', coding for the C terminus of the protein, with a BamHI site underlined.

The amplified gene product was digested with NdeI and BamHI before ligation into the pET-11a expression vector (Novagen), to generate the resulting plasmid pEH214. The DNA sequence of the gene was identical to that reported previously (5). The plasmid vector pEH214 was transformed into E. coli strain B834 (DE3), and an inoculum from a single colony was grown at 37 °C in a 2YT batch culture to an optical density at 600 nm (A₆₀₀) of 0.4. Expression was induced through the addition of 0.5 mM isopropyl-1-thio--D-galactopyranoside, and growth continued for a further 4 h. Cells were recovered by centrifugation (5000 rpm in a Sorvall GSA rotor at 4 °C for 30 min). The cell pellet was resuspended in a minimum volume of 20 mM Tris, 1 mM EDTA, 150 mM NaCl, 0.01% phenylmethylsulfonyl fluoride at pH 7.6 and stored frozen at 20 °C. Purification was by an adaptation of the method described by Talarico et al. (5) with the following modifications. A 220-ml DEAE-Sephacel column was used instead of Q Sepharose, the chromatofocusing step was omitted, and a 100-ml Sephacryl S-100 column was used for the final size fractionation step. The purity was estimated to be over 95% by SDS-polyacrylamide gel electrophoresis. The final preparation was stored at 4 °C at a concentration of 4 mg/ml, with the addition of 0.05% (w/v) sodium azide. The specific activity was 2.0 µmol/min/mg, and the final yield gave a total of 6 mg of pure HPPK per liter of culture. N-terminal sequencing of this protein product gave the sequence Thr, Val, Ala, Tyr, and the results of electrospray mass spectrometry gave a mass of 17,948 Da, showing that the N-terminal methionine residue had been removed.

Protein Determination-- Protein concentrations were measured using the method of Bradford (21).

Assay for 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase Activity-- HPPK activity was measured in 1 ml of 50 mM Hepes/NaOH buffer (pH 7.6) containing 10 mM MgCl₂, by coupling to a sequence of consecutive enzyme reactions in situ. The HPPK product, 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, was converted by an excess of DHPS, the next enzyme in the folate pathway, into dihydropteroate and inorganic pyrophosphate. The pyrophosphate is then converted to inorganic phosphate by cleavage with inorganic pyrophosphatase. Inorganic phosphate is used by purine nucleoside phosphorylase to cleave MESG into the products ribose-1-phosphate and a purine base. The cleavage of MESG gives an absorbance change at 360 nm between pH 6.5 and 8.5 (19). The reaction was initiated by the addition of 1.7 µg of purified HPPK in the presence of 0.015 units of purified DHPS from S. pneumoniae (22). The concentrations of other components were as described by Webb (19). The progress of the reaction was monitored in real time at 25 °C using a UVICON 922 Spectrophotometer (Kontron Instruments).

Equilibrium Fluorescence Titration-- Titrations were carried out by measuring the changes in fluorescence of either MANT-ATP or HMDP upon binding to HPPK. Measurements were made using a quartz cuvette containing 3 ml of 25 mM Hepes/NaOH (pH 7.5) plus 2.5 mM MgCl₂, maintained at 25 °C. The solution was stirred continuously during the course of the titration. Titration with HMDP required anaerobic conditions, which were obtained by purging the buffer extensively with oxygen-free nitrogen gas before the experiment and maintaining a steady stream of nitrogen across the surface of the solution during the titration measurements. For titrations conducted using MANT-ATP, the excitation wavelength was 356 nm, and the emission wavelength was 440 nm. For measurements based on HMDP fluorescence, the excitation wavelength was 330 nm and the emission wavelength was 430 nm. Fluorescence values were corrected for the inner filter effect using the method described previously (23).

Stopped Flow Fluorimetry-- Stopped flow measurements were carried out using an SX.18MV spectrometer (Applied Photophysics, Leatherhead, UK). The light source used was a 150-watt xenon lamp; for experiments using MANT-ATP, the excitation wavelength was 356 nm, using a 2.5-mm slit width. For HMDP, the excitation wavelength was at 330 nm, and a 3.0-mm slit width was used. The temperatures of the drive syringes and observation chamber were controlled by water circulation, and all experiments were carried out at 25 °C. The mixing ratio was 1:1 for all experiments. Solutions of ligands and enzyme were prepared by dilution into 25 mM Hepes/NaOH and 2.5 mM MgCl₂ (pH 7.5). The buffer was purged extensively with oxygen-free nitrogen gas immediately before use. Anaerobic conditions were maintained in the drive syringes by using an accessory, supplied by the manufacturer, that fitted over the seals of the drive syringes and was filled with a continuous stream of oxygen-free nitrogen gas. The flow tubing was purged of oxygen by presoaking in glucose oxidase and glucose, as recommended by the manufacturer. Finally, the water in the circulating water bath was purged of oxygen with oxygen-free nitrogen gas and treated with dithionite immediately before use. These precautions were sufficient to prevent HMDP oxidation during the lifetime of the experiment. Data acquisition and processing were controlled by a 32-bit RISC processor workstation. Apparent rate constants were determined by fitting the data to a single exponential expression using the Applied Photophysics SpectraKinetic software version 4.38.

Hummel and Dreyer Binding Studies-- A column of Sephadex G-50 (10-mm inner diameter × 300 mm) was connected to a fast protein liquid chromatography apparatus (Amersham Pharmacia Biotech) and equilibrated in 25 mM Hepes/NaOH and 2.5 mM MgCl₂ buffer at pH 7.5 at a flow rate of 0.5 ml/min. The column was then saturated with an appropriate ligand (5 µM AMP-CPP, 5 µM HMDP, or 10 µM ATP) before the addition of 0.1 ml (0.4 mg) of HPPK stock. The optical density of the eluent was monitored at 254 nm using an Amersham Pharmacia Biotech UV-MII detector.

Data Analysis-- Steady state reaction velocities were fitted using the software package Enzfitter (Biosoft, Cambridge, UK). The data were fitted using the Marquardt-Levenberg method with simple weighting.

For the titration of HPPK into MANT-ATP, the concentration of enzyme in the bound form was calculated from the quadratic root,

(Eq. 1)

where E_t is the total concentration of enzyme, A_t is the total concentration of MANT-ATP, and K_d is the equilibrium dissociation constant. Fluorescence values were then calculated from the expression,

(Eq. 2)

where F is the calculated fluorescence value, F_o is the starting fluorescence value (when [HPPK] = 0), and F_amp is the total amplitude of fluorescence change at infinite [HPPK]. The data were fitted using Enzfitter, employing the Marquardt-Levenberg method and with simple weighting.

For the experiments in which MANT-ATP is displaced from HPPK by another ligand, calculation of the fluorescence requires computation of the residual amount of enzyme·MANT-ATP binary complex that remains at any point during titration with the second ligand. This must be determined by solution of the following cubic,

(Eq. 3)

where [EA] is the concentration of the enzyme·MANT-ATP binary complex, and a, b, c, and d are defined as follows,

(Eq. 4)

(Eq. 5)

(Eq. 6)

(Eq. 7)

where E_t is the total concentration of enzyme, A_t is the total concentration of MANT-ATP, K_a is the equilibrium dissociation constant for MANT-ATP, K_b is the equilibrium dissociation constant for the competitor ligand (e.g. ATP), and B_t is the total concentration of competitor ligand. For each concentration of B_t, the value of [EA] was determined iteratively using Newton's method. Fluorescence values were then calculated from the expression F = F_o + F1·([EA]/[EA]_o), where F_o + F1 and [EA]_o are the fluorescence value and concentration of EA when B_t = 0, and F1 is a constant. Optimal values of K_b and F1 were then determined by minimization of the sum of the square of the residuals between the observed and calculated fluorescence values.

For the titration of HPPK into AMPCPP and HMDP, a compulsory order binding mechanism was assumed, with AMPCPP binding first, followed by HMDP to form the nonproductive HPPK·AMPCPP·HMDP ternary complex. There are therefore only two equilibria to consider: HPPK + AMPCPP = HPPK·AMPCPP and HPPK·AMPCPP + HMDP = HPPK·AMPCPP·HMDP. If ligand A represents AMPCPP and ligand B represents HMDP, the following can be shown,

(Eq. 8)

where

(Eq. 9)

and A_t is the total concentration of AMPCPP, B_t is the total concentration of HMDP, E_t is the total enzyme concentration, K_a is the dissociation constant for AMPCPP binding to HPPK, and K_b is the dissociation constant for HMDP binding to the HPPK·AMPCPP binary complex. For each concentration of E_t, the values of [EA] and [EAB] were determined iteratively using Newton's method. Fluorescence values were then calculated from the expression F = F_o + F1·([EAB]/B_t), where F_o is the fluorescence value when E_t = 0 and F1 is a constant. Optimal values of K_b and F1 were then determined by minimization of the sum of the square of the residuals between the observed and calculated fluorescence values.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition of HPPK by AMPCPP-- In order to study the binding of individual ligands to HPPK, it was useful to be able to generate a nonproductive ternary complex containing a substrate analogue. The nonhydrolyzable ATP analogue AMPCPP was identified as a potential inhibitor of HPPK, because it contains a carbon atom at the scissile bond for pyrophosphoryl transfer from ATP. A novel and convenient spectrophotometric assay for HPPK was developed that used the next enzyme in the pathway, DHPS, to form inorganic pyrophosphate from the products of the HPPK reaction. The pyrophosphate was then converted to phosphate with pyrophosphatase and linked to a phosphate-dependent colorimetric change using purine nucleoside phosphorylase (see "Experimental Procedures"). Measurement of the steady state velocity of the HPPK reaction at a range of different AMPCPP and ATP concentrations established that AMPCPP was a competitive inhibitor with respect to ATP, with a K_i of 0.31 µM (data not shown).

Hummel and Dreyer Measurements of Ligand Binding to HPPK-- The ligand binding characteristics of E. coli HPPK were examined using the method of Hummel and Dreyer (24). This is convenient to use, because both ATP and HMDP absorb ultraviolet light. A column of Sephadex G-50 gel matrix was pre-equilibrated with buffer containing the selected ligand and HPPK applied in a small volume. The absorbance of the eluent was monitored as a function of time. In the absence of any equilibrating ligand, HPPK eluted as a single absorbance peak (data for these experiments not shown). If the column was pre-equilibrated with ATP or AMPCPP, a peak of increased absorbance eluted that is coincident with the enzyme, which arose from the formation of the enzyme·ATP binary complex. This peak was followed by a trough of reduced absorbance, which arose from depletion of ligand by binding to the enzyme. The experiment was repeated using HMDP as the equilibrating ligand; the elution profile was identical to the control without ligand, indicating that the apoenzyme does not bind to HMDP. Inclusion of AMPCPP with HMDP in the equilibrating buffer gave a substantially increased absorbance peak coincident with HPPK elution, followed by a large trough, indicating that binding of both ligands occurs under these conditions. The results suggest a compulsory order of substrate binding, at least at the low concentrations of ligands studied here, with AMPCPP binding to the enzyme first, followed by HMDP. We proceeded to test this hypothesis by a more detailed analysis of ligand binding using fluorescence detection.

Binding of MANT-ATP-- 2'(3')-O-(N-Methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) is a fluorescent analogue of ATP that has been used to study the binding of ATP to other proteins (25, 26). Preliminary experiments established that MANT-ATP is a substrate for HPPK and would therefore be an ideal probe for ATP binding (data not shown). Titration of HPPK into a fixed concentration of MANT-ATP led to a 2.5-fold enhancement of fluorescence, and the data were consistent with a single, saturable binding site for MANT-ATP, with a K_d of 10.4 µM (Fig. 2). The binding constants for other ligands can be obtained by competition; for example, the addition of ATP to the HPPK·MANT-ATP binary complex reduced the fluorescence in a manner consistent with competitive displacement of MANT-ATP by ATP (Fig. 3). ATP and MANT-ATP were found to have similar equilibrium binding constants, but AMPCPP binds approximately 10-fold more tightly (Table I). A product of the reaction, AMP, binds about 10-fold more weakly than ATP, and, interestingly, the enzyme has affinity for GTP and GMP, albeit 75-fold weaker than for ATP binding.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   Fluorimetric titration of HPPK into MANT-ATP. Purified HPPK was titrated into a solution of 2 µM MANT-ATP. Other conditions are as described under "Experimental Procedures." Data points are means for three observations. The data were fitted to a model for a single, saturable binding site using the program Enzfitter. The graph shows the predicted fluorescence, in dotted lines, for a Kd value of 10.4 ± 0.3 µM with a fluorescence amplitude of 366 ± 7.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Displacement of bound MANT-ATP from HPPK by ATP. ATP was titrated into a solution containing 2 µM MANT-ATP and 3.5 µM HPPK, and the fluorescence was measured at the concentrations shown. Other conditions are as described under "Experimental Procedures." Data points are means for three observations ± S.E. The data were fitted to a model for competitive inhibition at a single binding site using a nonlinear least squares fitting method described under "Experimental Procedures." The graph shows the predicted fluorescence, in dotted lines, for a Kd value for ATP of 4.5 µM.

Binding of HMDP-- The fluorescence of the pterin ring can be exploited to measure the binding of HMDP to the enzyme. The addition of HPPK alone to HMDP resulted in no detectable change in pterin fluorescence, presumably because the apoenzyme does not bind to HMDP. The addition of HPPK to AMPCPP and HMDP, however, resulted in a concentration-dependent increase in fluorescence (Fig. 4). Similar results could be obtained if AMPCPP, rather than HPPK, was titrated into HMDP and HPPK (results not shown). The data in Fig. 4 were analyzed according to a compulsory order model where AMPCPP binds to the enzyme first, followed by HMDP; the predicted results are shown as dashed lines in Fig. 4 and gave an estimated K_d for the binding of HMDP to the HPPK·AMPCPP binary complex of 36 nM.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   HMDP fluorescence titration by HPPK. HPPK was titrated into a solution of 0.20 µM HMDP and 0.15 µM AMPCPP; other conditions are as described under "Experimental Procedures." Data points are means ± S.E. for three observations. The data were fitted to a sequential binding model, with AMPCPP binding first followed by HMDP, using a nonlinear least squares fitting method described under "Experimental Procedures." The graph shows the predicted fluorescence, in dotted lines, for a Kd value of 36 nM for HMDP.

Pre-steady State Kinetics-- The equilibrium measurements described above were extended to measure the rate constants for substrate binding to HPPK by stopped flow fluorimetry. Rapid mixing of HPPK and MANT-ATP resulted in a slow increase in fluorescence that can be fitted to a single exponential (Fig. 5). The apparent rate constant (k_app) was measured at a series of different MANT-ATP concentrations, and the results are shown in Fig. 6. The intercept gives an estimate of the rate constant for dissociation of MANT-ATP from the enzyme·MANT-ATP binary complex (k₁); in this case, it is 0.47 s¹. The gradient of the line gives a measure of the association rate constant (k₊₁) for MANT-ATP binding to HPPK of 3.9 × 10⁵ M¹ s¹.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Stopped flow fluorescence response of MANT-ATP following rapid mixing with HPPK. Syringe A contained 8.0 µM MANT-ATP, and syringe B contained 0.5 µM HPPK. The superimposed line shows a single exponential fit to the data, with an apparent rate constant of 2.1 s1.

View larger version (7K): [in this window] [in a new window]   Fig. 6.   Dependence of apparent rate constant for MANT-ATP binding to HPPK with MANT-ATP concentration. Data were collected as described under "Experimental Procedures." Each point is the mean ± S.E. for three observations. The final concentration of HPPK (after mixing) was 0.25 µM. The data were fitted by linear regression as implemented in Enzfitter to yield a gradient of 3.9 ± 0.3 × 105 M1 s1 and intercept of 0.47 ± 0.21 s1.

The fluorescence of the pterin ring was used to monitor the binding of HMDP to the enzyme. Rapid mixing of HMDP plus AMPCPP in syringe A with the apoenzyme in syringe B resulted in an increase in fluorescence that could be described by a single exponential, similar to that shown in Fig. 5 (data not shown). The same result was obtained if the enzyme and HMDP were pre-equilibrated in syringe B and mixed rapidly with AMPCPP in syringe A. If, however, the enzyme was pre-equilibrated with AMPCPP in syringe B and then mixed with HMDP in syringe A, the slow fluorescence response was not observed. A series of experiments were carried out using different concentrations of HMDP and AMPCPP in syringe A and a fixed concentration of HPPK in syringe B. If the HMDP concentration is fixed, the apparent rate constant (k_app) for fluorescence change varies in a linear manner with [AMPCPP] (Fig. 7). k_app did not vary, however, when the AMPCPP concentration was fixed and [HMDP] was changed up to 50 µM (data not shown). Our interpretation of these results is that the association of AMPCPP with the HPPK apoenzyme is slow and is followed by the relatively rapid addition of HMDP. The slow increase in HMDP fluorescence is therefore dependent on the rate of association of HPPK with AMPCPP, and as soon as the binary complex is formed, HMDP binds rapidly with a concomitant change in fluorescence. If this interpretation is correct, the gradient of the line in Fig. 7 is a measure of the association rate constant (k₊₁) for AMPCPP binding to HPPK; this value (1.05 × 10⁵ M¹ s¹) is the same order of magnitude as that obtained above for MANT-ATP (3.9 × 10⁵ M¹ s¹). The intercept of the line in Fig. 7 therefore corresponds to the dissociation rate constant for HMDP from the nonproductive ternary complex (k₂) and gives a value of 0.50 s¹.

View larger version (8K): [in this window] [in a new window]   Fig. 7.   Dependence of apparent rate constant for AMPCPP binding to HPPK with AMPCPP concentration. Data were collected as described under "Experimental Procedures." Each point is the mean ± S.E. for three observations. The final concentrations of HPPK and HMDP were 1.25 and 2.5 µM, respectively. The data were fitted by linear regression as implemented in Enzfitter to yield a gradient of 1.05 ± 0.01 × 105 M1 s1 and intercept of 0.50 ± 0.06 s1.

Steady State Kinetics-- Although some steady state kinetic data have been reported previously for HPPK, these studies were confined to measurements of apparent K_m values for one substrate at a fixed concentration of the second substrate. The results of the steady state kinetics of E. coli HPPK at multiple concentrations of ATP and HMDP were analyzed assuming the initial velocity for a compulsory order ternary complex reaction, which is given by Equation 10.

(Eq. 10)

This expression assumes that substrate A binds first to the enzyme, followed by substrate B (27).

The data were fitted to this expression using Enzfitter, to give the following estimated parameters: K_m^A(ATP) = 3.44 ± 0.02 µM; K_m^B(HMDP) = 0.60 ± 0.01 µM; K_i^A = 2 × 10⁷ ± 0.08 µM (steady state velocity data not shown). The K_i^A term is effectively zero within the limits of error, so the data can be fitted to the following simplified expression.

(Eq. 11)

Under these circumstances, the apparent K_m for each substrate is independent of the concentration of the second substrate. The data were in good agreement with Equation 11 and gave a K_M^A(ATP) of 3.2 ± 0.3 µM and a K_M^B(HMDP) of 0.68 ± 0.07 µM. The results show that the steady state kinetic data are at least consistent with a compulsory order ternary complex mechanism, within the limits of experimental error.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results presented here have provided evidence for a compulsory order of addition of the two substrates to HPPK, with ATP binding first in a slower step, followed by the more rapid addition of HMDP. It is important to try to reconcile these observations with the reported crystal structures of E. coli and H. influenzae HPPK in complex with various ligands. Xiao et al. examined the binding of ATP and HMDP to E. coli HPPK independently by NMR; both ligands induced significant changes in the backbone amide ¹H or ¹⁵N chemical shifts (15). It should be noted, however, that these experiments were conducted at millimolar concentrations of protein and ligand, and the binding of HMDP to the apoenzyme may be occurring at a much lower affinity than the high affinity binding to the AMPCPP·HPPK binary complex observed here. Stammers et al. (16) have reported the solution of two crystal structures with HMDP analogues bound to E. coli HPPK; these compounds, 40Y67 and 87Y76, contain methyl- or phenethyl- substituents at the 7-position within the pterin ring and were originally identified as potent inhibitors of HPPK by Wood and co-workers (28). It may be the case that 40Y67 and 87Y76 have higher affinity for the apoenzyme than HMDP itself, which would explain why observable electron density for the ligand can be seen in the binary complex with HPPK.

Hennig et al. (14) have reported the measurement of an equilibrium binding constant of 20 µM for ATP binding to H. influenzae HPPK, determined by titration of intrinsic tryptophan fluorescence. No observations were made, however, on the binding of ATP analogues, nor were the pre-steady state kinetics of binding investigated. We find here that MANT-ATP is a useful probe of ATP binding to HPPK; the addition of the fluorescent methylanthraniloyl group to the 2' or 3' group of the ribose ring does not seriously impair the binding affinity of MANT-ATP for HPPK, compared with ATP. Unfortunately, at the time of writing, only the coordinates of the E. coli apoenzyme form are available from the Protein Data Bank (accession code 1HKA). Most of the interactions with ATP observed in the complex with E. coli HPPK are apparently with the adenine ring or the triphosphate portions of the molecule, so it appears plausible that MANT-ATP could act as an effective analogue of ATP (16). Two magnesium ions are chelated within the complex by the phosphate oxygen atoms in ATP and also by Asp-95 and Asp-97 from the protein; removal of these interactions may explain why AMP binds with about 10-fold lower affinity than ATP.

The results of stopped flow experiments using MANT-ATP or AMPCPP give similar values for the on-rate for binding of both of these ligands to HPPK, of about 10⁵ M¹ s¹. This is approximately 3 orders of magnitude slower than the fastest second order rate constants for small ligands binding to proteins (29). A rough estimate of the rate constant for the addition of HMDP to the AMPCPP·enzyme binary complex can be obtained from the equilibrium binding constant (36 nM) and the measured off-rate for HMDP (0.50 s¹); the ratio gives a value of 1.4 × 10⁷ M¹ s¹. This suggests that AMPCPP and, by inference, ATP bind slowly to the enzyme, but once the binary complex is formed the addition of HMDP is very rapid. The very high binding affinity of the AMPCPP·HPPK complex for HMDP probably reflects the low concentration of this intermediate in vivo. It also suggests that the design of antimicrobial inhibitors, targeted at the HMDP binding site, should be based on the HPPK·ATP binary complex, rather than the apoenzyme.

Although the present study has illuminated some aspects of the HPPK reaction, there are still several details of the structure and mechanism of this enzyme that remain to be elucidated. The structural basis for the low affinity of the apoenzyme for HMDP and the high affinity of the AMPCPP·HPPK binary complex for the ligand is unclear at present; very few structural changes apparently occur upon formation of the 87Y76·ATP·HPPK ternary complex (16). The molecular basis for the catalysis of the pyrophosphoryl transfer is also uncertain, although it has been suggested that the magnesium ions, in conjunction with Asp-95 and Asp-97, may play a role in stabilizing the transition state. In general, the reaction mechanisms of pyrophosphokinases are poorly understood, but the extent to which HPPK is a paradigm for this type of enzyme remains to be seen.

	ACKNOWLEDGEMENTS

We are grateful to David Lascelles for technical assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Fellow of the Lister Institute of Preventive Medicine. To whom correspondence should be addressed: Dept. of Biomolecular Sciences, UMIST, Sackville St., Manchester M60 1QD, United Kingdom. Tel.: 44 161 200 4207; Fax: 44 161 236 0409; E-mail: Jeremy.Derrick@umist.ac.uk.

Published, JBC Papers in Press, April 5, 2000, DOI 10.1074/jbc.M000331200

	ABBREVIATIONS

The abbreviations used are: HPPK, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; DHPS, dihydropteroate synthase; HMDP, 6-hydroxymethyl-7,8-dihydropterin; DHPPP, 6-hydroxymethyl-7,8-dihydropterin pyrophosphate; MANT-ATP, 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate; AMPCPP, ,-methyleneadenosine triphosphate; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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