Eur. J. Biochem. 69, 195-201 (1976)

The Binding of Specific Ligands to Adenosine-Triphosphate Phosphoribosyltransferase Torbj$rn DALL-LARSEN and Leiv KLUNGSQYR Department of Physiology, University of Bergen (Received March 19/July 7, 1976)

Ligand binding by adenosine-triphosphate phosphoribosyltransferase was studied by different methods. 200 000 daltons of enzyme bound approximately 3 molecules of histidine cooperatively with a Hill plot slope of 1.23 (half-maximal binding at 520 pM). AMP increased the affinity of the enzyme for histidine (half-maximal binding at 80 pM). In the presence of AMP the binding of histidine was strongly cooperative with a Hill plot slope of 2.3. The transferase binds a little more than 3 molecules of AMP per hexamer of enzyme with a dissociation constant of the transferase . AMP complex of approximately 25 pM. ATP was able to displace radioactive AMP from the enzyme only at a concentration ratio of 25 in favour of ATP. The transferase bound 3 molecules of ATP per 200000 daltons to an inhomogeneous population of sites, or by a mechanism of negative cooperativity. The binding of phosphoribosyladenosine triphosphate took place preferably at 1-2 sites per hexamer of enzyme, depending on several factors including the magnesium concentration.

The biosynthesis of histidine in Escheviclzia coli is regulated on the enzyme level by several different mechanisms acting on the first enzyme in the pathway, phosphoribosyl-adenosine triphosphate : pyrophosphate phosphoribosyltransferase (ATP phosphoribosyltransferase). The mechanisms include end product (histidine) feedback inhibition [I, 21, energy charge control acting synergistically with end product inhibition [2], and first product inhibition by PRibATP which is also synergistic with the histidine inhibition ~ 3 1 . The inhibition by histidine of the phosphoribosyltransferase reaction is asymmetric, so that starting from certain concentrations of ATP, P-Rib-Pz, and PRibATP the reaction is strongly inhibited by histidine if pyrophosphatase is added so that more PRibATP is formed, and only slightly inhibited if PP; is added, so that ATP and P-Rib-P2 are formed [3]. A slightly substrate inhibition is observed when PRibATP is the substrate (Klungs$yr, unpublished observation). Klungsgyr and Atkinson [3] made preliminary binding studies with histidine, and obtained results which indicated that 200000 daltons of the enzyme bind 3 molecules of histidine. They also found that -~

Ahbreviations. PRibATP, N-1-(5-phosphoribosyl)-adenosine 5’-triphosphate; P-Rib-Pz, 5-phospho-a-D-ribose I-diphosphate. Enzjmr. ATP phosphoribosyltransferase from Esclwrichia coli or Wl-( 5’-phosphoribosyl)-ATP : pyrophosphate phosphoribosyltransferase (EC 2.4.2.17).

AMP and PRibATP increased the affinity of the transferase for histidine, and that AMP binds to the enzyme. More information was necessary about the binding of the transferase to its specific ligands, and binding studies were carried out with histidine, AMP, ATP and PRibATP. Parts of the data presented here have been published previously in an abbreviated form [4].

MATERIALS AND METHODS ATP phosphoribosyltransferase was prepared as described by Klungs$yr and Atkinson [3], and carried through the second dialysis step. The enzyme was then dissolved in basal buffer (see below) 0.1 M NaCl IS], and dialyzed against that solvent in the rapid dialysis device [3] for 3 h before use. No histidase activity was observed in the purified enzyme preparation. ‘‘C-labelled histidine, AMP and ATP were purchased from The Radiochemical Centre (Amersham, England). Labelled PRibATP was prepared from labelled ATP with an excess of P-Rib-Pz by the method of Smith and Ames [6,7], and purified as described by these authors on an ion-exchange column with a lithium chloride gradient. Unlabelled PRibATP was prepared in the same way, with excess ATP. Repeated ion-exchange chromatography yielded products of satisfactory purity, as judged from absorption spectra [7], and from the decrease in absorbance at

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Fig. 1. The binding of histidine I O A T P phosphoribosyltransferase measured by equilibrium dialysis. (A) The enzyme and [14C]histidinewere dissolved in basal buffer with 0.1 M NaCI. The cells were rotated at 3 rev./min in the cold room (2-4°C) for 16 h. Standard error and number of measurements are shown in the figure. Five different enzyme preparations were used, the protein concentrations were: 2.6, 3.6, 4.95, 5.9 and 8.15 mg/ml. n is defined as the number of molecules of ligand bound by 200000 daltons of' enzyme, c is the ligand concentration (B) Scatchard plot of the average values in (A)

290 nm at pH 8.5 in the presence of pyrophosphate and transferase. ATP, AMP and histidinol were purchased from Sigma Chemical Co., St. Louis. Radioactivity assays were carried out in the scintillation solvent described by Kuehn et al. [8], and the samples were counted in Beckman or Nuclear Chicago liquid scintillation counters. Protein was determined by the method of Klungs$yr [9]. Binding Procedures Binding studies were carried out by several different methods, such as the rate of dialysis method of Colowick and Womack [lo], equilibrium dialysis on a micro scale [ll], and the gel filtration method of Hummel and Dreyer [12]. All binding experiments were carried out in 10 mM imidazole buffer, pH 7.2, containing 0.5 ml mercaptoethanol per 1 (basal buffer [ 5 ] ) and 0.1 M NaCI. The dialysis membranes were from Union Carbide Corporation (no. 8). The membranes were stretched over night before use by filling them with water and keeping the tubing in vacuum with the lumen open to atmospheric pressure. For equilibrium dialysis the stretched membranes were further treated as described by Englund et al. [ll]. RESULTS Experiments with Histidine An attempt was made to study the binding of histidine by the dialysis rate method [lo]. After ad-

dition of the radioactive ligand the rate of elution of tracer increased to the level of that seen with a control without enzyme, and remained there even after addition of high concentrations of histidine. We have explained the failure of the method with this ligand by the fact that histidine is bound in a cooperative manner to the transferase. At low concentrations of histidine, therefore, the enzyme has little affinity for the ligand. The method is based on the assumption that a large fraction of the initially added trace amount of radioactive ligand is bound and this does not apply in the case of histidine. (From data obtained by equilibrium dialysis experiments, the radioactive histidine which was free to dialyze was calculated for several histidine concentrations in a simulated dialysis rate experiment. It could be shown that the rate of dialysis of radioactive ligand would reach a maximum at the lowest concentrations, and that addition of nonradioactive histidine would not increase the dialysis rate. This may also be realized from the shapes of the Scatchard plots in Fig. 1 and 2. At the lowest histidine concentrations the ratio between bound and free histidine is very low, in contrast to the requirements of the dialysis rate method.) The microdialysis method [ l l ] proved suitable for binding studies with histidine. Experiments were made with five different enzyme preparations, in the absence and the presence of 500 pM AMP. The results are presented in Fig. 1 and 2. The highly curved Scatchard plot [13] of Fig.2 shows a strong positive cooperativity, and when the results are arranged in a Hill plot the maximum slope of the line is 2.3. 200000 daltons of enzyme bound 2.9 molecules of histidine.

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Fig. 2. The binding of histidine lo A T P phos[~hol.ihos)..ltrunsfcrasrin ilirpresence OJ'0.5 m M A M P . (A) The experiments were run in parallel with the experiments in Fig. 1, the only difference being the addition of AMP. Standard error and the number of measurements are shown in the figure. Five enzyme preparations were used with the same protein concentrations as in Fig. 1. n and c are defined as in Fig. 1. (B) Scatchard plot or the average values in (A)

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Fig. 3. Scalehard plot of' data fiw A M P binding to ATP phosphorihos},ltransferuse measured hy the rate of' dialysis method (101 at 2- 4 'C.The upper chamber initially contained the enzyme and trace amounts of ''C-labelled A M P dissolved in basal buffer with 0.1 M NaCI. The lower chamber was perfused with the same buffer. Unlabelled A M P was added to the upper chamber to give the desired concentrations. n and c are defined as in Fig. 1. The protein concentration in the upper chamber at the beginning of the experiment was 5 . 2 mg/ml

The dialysis rate method [lo] gave good results when AMP was the ligand. A typical experiment is presented in Fig. 3. There is no obvious deviation from linearity in the Scatchard plot, and in the experiment shown 3.4 molecules of AMP were bound per 200000 daltons of the enzyme. Experiments were also performed with the microdialysis method [ll], and the gel filtration method of Hummel and Dreyer [I 21, and similar results were obtained [4]. The average value of n from four different enzyme preparations with three different methods was 3.35 molecules of AMP per 200000 daltons of enzyme. The Kd was 25 pM. From previous studies [3,5,14,15] it appeared that the effect of AMP was biphasic. Concentrations around 50 pM were effective to increase the affinity of the enzyme for histidine as demonstrated kinetically [3] and by sedimentation in the ultracentrifuge [5].This high-affinity site for AMP was further characterized in the present binding experiment. With the dialysis rate method [lo] it could be shown that the preference for AMP was considerable, since high concentrations of ATP were needed to displace AMP (Fig. 4). Experiments with ATP When ATP was used as the ligand for the synthetase in the dialysis rate method [lo], an anomaly was observed which was different from that seen with

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Fig. 5. Binding of A T P by A T P phosplzorihosyltran.~~~ruse nieasured by eyuilihrium dialysis ut 2- 4°C with [I4C]ATP in the presence of 1 m M MgCIz. (A) Conditions as in Fig. 1. Standard errors and number of measurements are given in the figure. Five different enzyme preparations were used, the protein concentrations were: 4.95, 5.88, 10.8, 10.8 and 12.6 mg/ml. n and c are defined as in Fig. 1. (B) Scatchard plot of the average values in (A)

histidine. After addition of radioactive ATP to the upper chamber in the presence of enzyme, the radioactivity eluted was less than expected. When large amounts of nonradioactive ATP were added, no increase in the elution of radioactive ATP was observed. The binding of ATP was therefore studied with the equilibrium dialysis method Ell]. It has been shown previously that the enzyme itself needs Mg2+ for its activity [14], but it has not been decided whether ATP is bound as the magnesium complex. For this reason we tested the binding of ATP in the regular binding

buffer (see Materials and Methods) and with 1 mM Mg2+ added to the buffer. Very little difference was observed between the experiments with or without Mg2'. The results of the experiments with Mg2+ present are shown in Fig.5. Five different enzyme preparations were used. Experiments with PRibATP

For binding studies with PRibATP equilibrium dialysis gave the best results. As previously published

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Fig. 6. The binding of PRihATP to ATP phos~~horihos~ltransferase studied hj) equilibrium dialysis at 2 - 4 "C with 3H-labelled PRibATP in the absence of tnagnesium. (A) Nine enzyme preparations were used with protein concentrations between 2.9 and 9.9 mg/ml. For measurements with a free ligand concentration above IS0 pM the protein concentration was doubled. The number of determinations and the standard errors are given in the figure. Conditions otherwise as in Fig. 1. n and c are defined as in Fig. 1. (B) Scatchard plot of the average values in (A)

Klungs$yr and Atkinson [ 3 ] demonstrated that the ratio between PPi and Mg2+ was important for the affinity of the transferase for PRibATP in the reversed reaction. When excess MgZ+ was present, the substrate-disappearance curve showed decrease in velocity Concentration n at at much higher PRibATP concentrations than when ~ _ _ _ ~ ~. ~____ PRibATP PPi was added in excess of Mg2+. It appears from 2-4°C 20 "C Fig.6 and 7 that Mg2+ was important also for the binding of PRibATP to the transferase. In the presPM ence of 1 mM Mg" the enzyme had less affinity for so 0.69 0.09 (4) 0.66 k 0.05 (4) the ligand, but more ligand was bound before a 0.29 f 0.24 (4) 200 0.47 & 0.34 (4) plateau was reached. Without MgZ+ one site was bound with high affinity, and then further binding took place much less readily. [4] we obtained a maximum in the PRibATP binding curve in many experiments. Searching for explanations we found that there was no appreciable decrease in DISCUSSION ligand concentration during dialysis, and that the Histidine is bound to ATP phosphoribosyltransenzyme retained its activity after completion of the ferase by a cooperative mechanism, with a maximum dialysis procedure. The appearance of a maximum in of 3 mol of histidine bound per mol (hexamer) of the binding curve is not only a function of the length enzyme. The present results were obtained by the of contact between enzyme and ligand as we originally equilibrium dialysis method which means prolonged believed [4].We are unable to explain the variable contact between ligand and enzyme, but were in good behaviour of the enzyme, since our preparation agreement with earlier published preliminary results [ 3 ] procedure was not consciously changed. In the enzyme obtained by the Hummel-Dreyer technique [12] and preparation where a maximum in the binding curve short-time contact between ligand and enzyme. Even is seen (Table l), a precipitate forms in the cells with if we can not demonstrate histidase activity in our the highest ligand concentrations where the binding enzyme preparations, the agreement between the two is low. This precipitate retains its enzymic activity, methods for obtaining binding data is additional proof but requires a period of preincubation after dilution that the ligand remains unchanged during prolonged with PRibATP-free buffer before full activity is redialysis. AMP increased the affinity of the transferase covered.

Table 1 . Number of motes PRihATP hound per 200000 daltons ATP plzosphoribosyltransferase ar low) and high ligand concentrations n = moles of bound PRibATP/mole protein. Protein concentration was based on a molecular weight of 200000. Results are given +_ S.E. with number of observations in parentheses

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Fig. I. The binding ofPRihATP to ATPphospkoribos~.ltransJerasein the presence ($1 mM M g s o 4 . (A) The conditions were the same as in Fig.6 except for the presence of MgS04 and that five different enzyme preparations were used with protein concentrations between 5.7 and 10.5 mg/ml. Standard errors and number of measurements are shown in the figure. n and c a r e defined as in Fig. 1. (B) Scatchard plot of the average values in (A)

for histidine without changing the number of molecules of histidine bound to the enzyme. The mechanism by which AMP increases the affinity of the enzyme for histidine is not clear. Both ligands stabilized the 8.9-S species in the ultracentrifuge [ 5 ] , but kinetic studies [3] and experiments with tritium exchange and aminonaphthalene sulfonate fluorescence [ 151 show that the hexamers stabilized by the two ligands are different. It is, however, possible that hexamer formation by AMP aids in the binding of histidine. Further conformational changes must then take place in connection with histidine binding which is cooperative (Fig. 2). The binding data for AMP clearly define a class of binding sites with a little more than three moles of ligand per hexameric enzyme, and a dissociation constant of 25 pM. It is also established [4] that 1 : N 6 etheno-adenosine 5'-monophosphate (FAMP) [16] binds to the same sites with a dissociation constant of 50 pM. In the absence of histidine the filling of these sites results in little inhibition of the transferase reaction with either &[4] or AMP [3,14]. Higher concentrations of & are strongly inhibitory in apparent competition with ATP [4] (and HBkon Kryvi, unpublished results). However, in a system as complex as this, competitive kinetics are no proof of direct competition between the substrate and the inhibitor at the substrate site. In kinetic experiments AMP alone displays negligible inhibition of the transferase around 500 pM. Therefore, if binding in addition to the already filled high-affinity sites takes place, this must happen without much interference in the binding of substrate ATP.

Evidence for kinetically undetectable, low-affinity sites for AMP, analogous with the inhibitory CAMP includes the following points. (a) The Scatchard plots for both AMP and &extrapolate to a little more than 3 sites per hexameric enzyme [4],indicating that the binding of both ligands may be inhomogeneous. (b) Enhancement of inhibition by histidine of the transferase at moderate histidine concentrations is much stronger at 400 pM AMP than at 100 pM even if the latter concentration is 4 times the Kd for binding of AMP at the high-affinity sites (Fig. 5 in [3]). The demonstrated competition by high concentrations of ATP with AMP (Fig. 4) probably takes place at the high-affinity AMP sites, because nearly 500 pM ATP was necessary to give detectable displacement of an AMP concentration of less than 20 pM. Magnesium ions had little influence on the binding of ATP. It therefore seems likely that the enzyme does not require Mg-ATP as a substrate. The Scatchard plot of the binding data for ATP was non-linear, and indicates either several classes of sites with different affinity for ATP, or negative cooperativity among identical sites. We have previously noted that high concentrations of ATP create an 8 . 9 3 form of the synthetase [5]and believe that these forms are inhibited states of the enzyme. In kinetic experiments we have also demonstrated direct inhibition of the transferase reaction by ATP at high enzyme concentrations. Bell and Koshland [17] reported negatively cooperative kinetics with ATP for the enzyme from Salmonella typhimuvium. The binding experiments with PRibATP (and to some extent ATP) were difficult and gave variable

20 1

T. Dall-Larsen and L. Klungs@yI

results. All experiments were carried out on ligands of satisfactory purity. The enzyme purification procedure [3] twice utilizes the changed solubility in ammonium sulfate in the presence of histidine. An enzyme is routinely obtained which is homogeneous in the ultracentrifuge in the presence of histidine and AMP [ 5 ] . Furthermore the enzyme yields well defined tryptic fingerprint maps with the expected number of spots [18]. The variability observed with PRibATP is not seen with histidine and AMP as ligands, which consistently yield reproducible results. We therefore believe that variations in PRibATP binding reflect subtle changes which can not be observed by standard kinetic methods or binding of the 'easy' ligands, histidine and AMP. At present we can only conclude that some enzyme preparations become reversibly denatured in the presence of high concentrations of PRibATP, and at the same time loose the ability to bind or retain PRibATP (Table 1). We are unable to predict which preparations will behave in this manner. Experiments carried out in the absence of Mg2+ extrapolate to one mole of PRibATP per hexameric enzyme, with a dissociation constant of 24 pM. Binding in excess of one site proceeded with much lower affinity. Kinetic experiments with high pyrophosphate, low Mg2+ gave an [S]o.5 of 3 pM for PRibATP in the reverse direction of the reaction [3]. Dimers liganded with PRibATP may apparently capture two additional unliganded dimers, and by doing so (sterically?) impede the binding of more PRibATP, and the dissociation of the bound group (product inhibition). The sticky dimer arises when preformed PRibATP is offered as a ligand, or by a conformational change during the catalytic process when PRibATP is formed [15]. The Mg2+ complex of PRibATP apparently has less affinity to the enzyme than the free product (a finding which is supported by kinetic data [3]). Also in this case the binding levels off at less than three moles of ligand per hexameric enzyme. Despite the variability of the results in the PRibATP binding studies, all results support the conclusion

that binding of PRibATP introduces restaints in the enzyme . ligand complex. These restraints seem to hinder binding and release of the product of the ATP phosphoribosyltransferase reaction. One of us (T. D.-L.) is supported by the Royal Norwegian Council for Science and the Humanities, Trine Tydal is thanked O r excellent technical assistance. Dr D. h. Atkinson gave valuable advice during the preparation of the manuscript.

REFERENCES 1. O'Donovan, G. G. & Ingraham, J. L. (1965) €'roc. .Wut/ Acrrrl. Sci. U . S . A .54, 451 -457. 2. Klungspyr, L., Hageman, J . H., Fall, L. & Atkinson, D. E. (1968) Biochemi~try,7, 4035 - 4040. 3. Klungsdyr, L. & Atkinson, D. E. (1970) BiochemistrJ; 9? 2021 -2027. 4. Klungs$yr, L., Kryvi, H. & Dall-Larsen, T. (1975) in S J ~ I ~ O sium on Mechanlsm of' Action and Rqulution 01' EKJW?P.Y (Holzer, H. & Keleti, T., eds) pp, 91 -96, Budapest. 5. Klungsgyr, L. & Kryvi, H. (1971) Biochim. Bioj7h.y.s. Acto, 227, 327- 336. 6. Ames, B. N., Martin, K. G. & G a r y , B. J . (1961)J. Biol. Cher77. 236, 2019-2026. 7. Smith, D. W. E. & Amcs, B. N . (1965) J . B i d . ('hem. 240, 3056- 3063. 8. Kuehn, G. D., Barnes, L. D. & Atkinson, D. E. (1971) Biochmntistry,10, 3945 - 3951. 9. Klungsgyr, L. (1929) A n d . Biochern. 27, 91 -98. 10. Colowick, S. P. & Womack, F. C. (1969) J . B i d . C h c m 244, 774- 771. 11. Englund, P. T., Huberman, J . A , , Jovin, T. M. & Kornberg, A. (1969) J . Biol. ('hem. 244, 3038-3044. 12. Hurnmel, J. P. & Dreyer, W. J. (1962) Bioc,him. Biophys. Artu, 63, 530-532. 13. Scatchard, G. (1949) Ann. N . Y . Acud. Sci. 51, 660-672. 14. Kryvi, H. & Klungsgyr, L. (3971) Biockim. Biophys. Actu, 23.5, 429-434. 15. Klungs$yr, L. (1971) Biwhemistry, 10, 4875-4880. 16. Barrio, J. R., Secrist, J. A . 111, Chien, Y.-H., Taylor, P. J., R o binson, J. L. & Leonard, N. J. (1973) FEBS Lctt. 2Y, 215218. 17. Bell, R. M. & Koshland, D. E., J r (1971) Bioorg. Chem. I . 409- 423. 18. Dall-Larsen, T., Fasold, H., Klungsdyr, L., Kryvi, H . , Meyer, C. & Ortdnderl, F. (1975) Eur. J . Biochem. 60, 103- 107.

T. Dall-Larsen and L. Klungsgyr, Fysiologisk Institutt, Universitet i Bergen. Arstadveien 19, N-5000 Bergen, Norway