Purification and characterization of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and comparison with the human enzyme

Molecular and Biochemical Parasitology 98 (1999) 29 – 41

Purification and characterization of Plasmodium falciparum hypoxanthine – guanine – xanthine phosphoribosyltransferase and comparison with the human enzyme Dianne T. Keough a, Ai-Lin Ng a, Donald J. Winzor a, Bryan T. Emmerson b, John de Jersey a,* a

Centre for Protein Structure Function and Engineering, Department of Biochemistry, Uni6ersity of Queensland, Brisbane, 4072, Australia b Department of Medicine, Princess Alexandra Hospital, Uni6ersity of Queensland, Brisbane, 4102, Australia Received 20 April 1998; received in revised form 15 September 1998; accepted 22 September 1998

Abstract The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides. Hypoxanthine– guanine – xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P. falciparum, making it a potential target for chemotherapy. Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage. Other groups have tried to express recombinant P. falciparum HGXPRT but only small amounts of activity were obtained. The successful expression of recombinant P. falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities. The measured molecular mass of 26 229 9 2 Da is in excellent agreement with the calculated value of 26 232 Da. A method to stabilise the activity and to reactivate inactive samples has been developed. The subunit structure of P. falciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KCl. Kinetic constants were determined for 5-phospho-a-D-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol. Differences in specificity between the purified P. falciparum HGXPRT and human hypoxanthine–guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphoribosyltransferase; Malaria; Plasmodium falciparum; Recombinant enzymes; Purine salvage

Abbre6iations: DEAE, diethylaminoethyl; DTT, dithiothreitol; HGPRT, hypoxanthine – guanine phosphoribosyltransferase; HGXPRT, hypoxanthine – guanine–xanthine phosphoribosyltransferase; Hx, hypoxanthine; PMSF, phenylmethanesulfonyl fluoride; PPi, pyrophosphate; PRib-PP, 5-phospho-a-D-ribosyl-1-pyrophosphate. * Corresponding author. Tel.: + 61-7-33654622; fax: + 61-7-33654699; e-mail: [email protected]. 0166-6851/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 8 ) 0 0 1 3 9 - X

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1. Introduction Malaria is estimated to kill 1.5 – 2.7 million people per year [1]. The most important of the parasites responsible for this disease is Plasmodium falciparum. Because of the increased prevalence of resistance of the parasite to drugs currently in use, there is an urgent need to develop new chemotherapeutic agents. The enzymes of purine and pyrimidine nucleotide synthesis have been suggested as potential targets for antiparasitic drugs [2]. In particular, purine salvage enzymes have been considered since protozoan parasites including P. falciparum are incapable of de no6o synthesis of purine nucleotides [2,3]. Hypoxanthine – guanine phosphoribosyltransferase (HGPRT; E.C. 2.4.2.8) salvages the purine bases, guanine and hypoxanthine Hx. It catalyses the synthesis of the 6-oxopurine mononucleotides, IMP and GMP, by transferring the nitrogen base to the 1-b-position of the ribose ring of 5-phospho-a-D-ribosyl1-pyrophosphate (PRib-PP) concomitant with the release of the pyrophosphate (PPi) moiety. This reaction has an absolute requirement for the presence of a divalent metal ion. In humans and other mammals, purine nucleotides can be synthesized by both de no6o and salvage pathways. P. falciparum homogenates have been found to contain high levels of hypoxanthine–guanine – xanthine phosphoribosyltransferase (HGXPRT) activity [3], indicating that HGXPRT plays a major role in purine salvage. Based on this knowledge, it is reasonable to propose that drugs designed to specifically target P. falciparum HGXPRT could prove to be valuable chemotherapeutic agents. Such drugs could potentially act by two mechanisms: (i) as inhibitors of the P. falciparum enzyme; and (ii) as substrates which, after conversion to their respective mononucleotides by HGXPRT, would inhibit other enzymes and/or be incorporated into parasite DNA or RNA resulting in eventual cell death [2]. The goal of developing selective inhibitors or toxic alternative substrates for the Plasmodium enzyme which have little or no effect on the human enzyme depends on a detailed knowledge of the active sites of the human and parasite enzymes.

Queen et al. [4] reported the partial purification of HGXPRT from P. falciparum grown in culture. Their studies were seriously hampered by the difficulty of growing large quantities of the parasite. However, the largest hurdle faced was the extreme instability of the enzyme activity during purification (e.g. loss of : 50% in 20 h). In attempts to overcome the first problem, several groups have reported the expression of recombinant P. falciparum HGXPRT in Escherichia coli [5,6]. However, very low activities were found in cell homogenates— far too little to attempt purification of the enzyme. In the present study, cDNA coding for P. falciparum HGXPRT has been cloned into the expression vector previously used in our laboratory to express human HGPRT [7]. Recombinant P. falciparum HGXPRT was purified to homogeneity and a procedure developed to stabilise its activity. A comparison of the properties of the P. falciparum and human enzymes, showing significant differences in substrate specificity, is reported.

2. Materials and methods

2.1. Chemicals, materials, and reagents The cDNA coding for P. falciparum HGXPRT was a gift from Dr R. Coppel, Walter and Eliza Hall Institute, Australia. PRib-PP was purchased from Sigma or from Fluka. The purine substrates were obtained from Sigma. Diethylaminoethyl (DEAE)-cellulose was a product of the James River Corporation. DNAase (1500 units ml − 1) was obtained from Promega. HgSepharose resin was synthesized by the method of Zappia et al. [8]. GMP-Sepharose was synthesized by the method of Hughes et al. [9]. The reversed phase column used for the separation of purine bases from their product mononucleotides was a Waters mBondapak (particle size: 10 mm; dimensions: 3.9 × 300 mm) fitted with an Uptight filter purchased from Activon.

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2.2. Cloning and sequencing of the Plasmodium falciparum hypoxanthine– guanine – xanthine phosphoribosyltransferase and human hypoxanthine–guanine phosphoribosyltransferase genes P. falciparum and human cDNA were cloned into the pT7-7 expression vector as previously described for human HGPRT [7]. Three single mutants of recombinant human HGPRT (C22A, C105A, and C205A) were prepared by splicing by overlap extension [10]. Selective digestion with restriction enzymes of the cDNA coding for these mutants and subsequent ligation resulted in the construction of a plasmid containing the cDNA coding for a mutant form of human HGPRT which contained all three amino acid substitutions, C22A, C105A, C205A [11]. The thiol residues were replaced by alanine to reduce the possibility of oxidation which occurs on storage of the wild type enzyme [12]. The plasmids were transformed into SF606 (ara, Dpro-gpt-lac, thi, hpt, F − ) E. coli cells [13]. Sequencing of the P. falciparum HGXPRT and human HGPRT cDNA was performed by automatic methods at the University of Queensland sequence analysis facility.

2.3. Expression P. falciparum HGXPRT and human HGPRT were expressed as described by Free et al. [7]. After expression and centrifugation, the cell pellets were suspended in two different buffer systems. For P. falciparum HGXPRT, the buffer used was 0.01 M phosphate, pH 6.9, containing 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), 200 mM PRib-PP, while, for the human enzyme, the buffer was 0.05 M Tris–HCl, 0.01 M MgCl2, 1 mM DTT, 1 mM PMSF, 200 mM PRib-PP, pH 7.4.

2.4. Enzyme acti6ity The enzyme activities for the purine bases guanine, Hx and xanthine were measured in a continuous spectrophotometric assay [14] at 25°C unless otherwise specified. Assay buffers were 0.1 M Tris –HCl, 0.11 M MgCl2, pH 8.5 or 0.1 M Tris –

31

HCl, 12 mM MgCl2, pH 7.45. Stock solutions of PRib-PP were prepared in water. The purine bases were also dissolved in water with the addition of a small amount of NaOH to solubilize them. The standard assay contained 1 ml of the assay buffer, 50 ml of 20 mM PRib-PP, 30 ml of 2 mM purine base and a suitable aliquot of enzyme. The Do values measured under assay conditions for guanine, Hx and xanthine reactions (pH 8.5) at 257.5, 245, and 255 nm were 5817, 2283, and 4685 M − 1 cm − 1, respectively. For guanine and Hx, the Do values were the same at pH 7.4; however, at pH 7.4, the Do for xanthine was 3794 M − 1 cm − 1. One unit of activity is defined as the number of mmol of purine base converted to its product mononucleotide min − 1.

2.5. Determination of protein concentration The protein concentration of purified P. falciparum HGXPRT was determined using an A 1% 1 cm of 8.0 while the A 1% 1 cm for the human enzyme was 5.3. These values were calculated by the method of Gill and von Hippel [15]. Xu et al. [16] have recently reported an A 1% 1 cm of 10 for the human enzyme.

2.6. Purification of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and human hypoxanthine–guanine phosphoribosyltransferase In both instances, E. coli cells were lysed as described by Free et al. [7]. Glycerol was added to the P. falciparum cell paste to give a final concentration of 10% (v/v). Two methods were then used to purify the P. falciparum enzyme to homogeneity: (i) the lysate DNA from 50 ml of cell paste (2 l of cell culture) was removed by hydrolysis by DNAase (1 ml ml − 1 of cell paste) in the presence of 10 mM CaCl2, followed by precipitation with protamine sulfate (final concentration, 1.5 mg ml − 1 of cell paste). After centrifugation, the enzyme solution was dialysed against 0.01 M phosphate, pH 7.1, for 2 h prior to loading onto the Hg-Sepharose column (4.3×1.7 cm) equilibrated in the same buffer. P. falciparum HGXPRT was eluted with this buffer containing 10 mM DTT;

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(ii) cell lysate from 50 ml of cell paste was loaded onto a DEAE-cellulose column (43× 2.2 cm) equilibrated in 0.01 M phosphate, pH 6.9. Under these conditions, the enzyme did not bind to the resin. Fractions containing P. falciparum HGXPRT were identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) since all activity was lost during the chromatography. Pooled fractions were loaded onto Hg-Sepharose and eluted as outlined in (i). Human HGPRT (C22A, C105A, C205A) was purified by batch DEAE-cellulose and GMP-Separose chromatography, using the same procedure as for the purification of wild type recombinant human HGPRT [12].

2.7. SDS-Polyacrylamide gel electrophoresis Enzyme samples were denatured and run on SDS-PAGE (12.5% gels) according to the method of Laemmli [17].

2.8. Mass spectrometry Mass spectrometry was performed using a PE SCIEX API 165 single quadrupole mass spectrometer equipped with an Ionspray atmospheric pressure ionization source, used in the positive mode. Full scan data were acquired by scanning from m/z 1000 –2400 with a scan step size of 0.1 Da and a dwell time of 3 ms. P. falciparum HGXPRT and human HGPRT were prepared by dialysis into 0.1% CH3COOH. The concentration of protein was : 0.5 mg ml − 1.

2.9. Determination of kinetic constants Kinetic constants were determined under the same conditions as described above for measuring enzyme activity. To measure Km values for PRibPP, the range of substrate concentrations was 12 – 1600 mM while, for guanine and Hx, the range was 2–60 mM. Initial data suggested that the Km for xanthine was significantly higher than that for guanine and Hx. Because of its low solubility, stock solutions of xanthine were prepared directly in the assay buffer. This allowed the range of substrate concentrations used to be 31 – 544 mM. The specific activity and Km value for allopurinol

were determined in 0.1 M Tris–HCl, 0.11 M MgCl2, pH 8.5 at 273 nm. The Do value for the reaction was 1700 M − 1 cm − 1 [18]. Kinetic data were analysed using a non-linear regression analysis program [19].

2.10. Separation of bases and mononucleotides by high performance liquid chromatography Purine bases were separated from their respective mononucleotides by high performance liquid chromatography (HPLC) according to the method of Wynants and Van Belle [20]. Stock solutions of the purine base (2 mM) were diluted by adding 30 ml to 1 ml of 0.01 M Tris–HCl, 12 mM MgCl2, pH 7.45. To a 200 ml aliquot of this solution was added 10 ml of 20 mM PRib-PP and 5 ml of the Plasmodium enzyme (3649 units ml − 1, measured at pH 8.5, guanine as the purine substrate) or 2.5 ml of the human enzyme (7633 units ml − 1 measured at pH 8.5, guanine as the purine substrate). The final concentrations of base and PRib-PP in the reaction mixture were 54 mM and 930 mM, respectively. Aliquots of the reaction mixture (50 ml) were injected onto the HPLC column and the eluant monitored at 245 nm.

2.11. Inhibition by mercuric chloride P. falciparum HGXPRT was dialysed into 0.01 M phosphate, 1 mM DTT, pH 7.1. The enzyme activity was measured at pH 8.5 with guanine as the purine substrate. To an aliquot of the enzyme, HgCl2 was added to give a final concentration of 3.2 mM and the activity of the enzyme determined after 3 min incubation on ice. The effect of HgCl2 on the activity of human HGPRT was determined in a similar experiment to that described for the parasite enzyme. In this case, the enzyme was first dialysed into 0.01 M phosphate, 0.01 M MgCl2, 1 mM DTT, pH 7.1. The enzyme activity was measured at pH 7.4 with guanine as the purine substrate.

2.12. Effect of KCl P. falciparum HGXPRT was dialysed into 0.01 M phosphate, 1 mM DTT, pH 7.1, containing 60

D.T. Keough et al. / Molecular and Biochemical Parasitology 98 (1999) 29–41

mM Hx and 200 mM PPi or into 0.01 M phosphate, 1 mM DTT, pH 7.1. Enzyme activity was measured before and after dialysis. Aliquots of 4.4 M KCl were added to the enzyme so that the final concentration of KCl was either 1.5 or 0.74 M. To separate solutions of enzyme, 7.5 ml of 13 mM PRib-PP were added (final concentration, 1.1 mM), immediately prior to the addition of the KCl. This experiment was repeated with human HGPRT except that, in this instance, the enzyme was dialysed into 0.01 M phosphate, 0.01 M MgCl2, 1 mM DTT, pH 7.1, prior to the addition of 4.4 M KCl in the presence and absence of PRib-PP. In this case, the final concentration of KCl was 1.2 M and the concentration of PRibPP, 1.3 mM.

2.13. Sedimentation equilibrium studies in the presence and absence of KCl Purified P. falciparum HGXPRT (pooled fractions from Hg-Sepharose; method ii) was dialysed into 0.01 M phosphate, 60 mM Hx, 5 mM DTT, pH 6.9 for 4 h at 4°C. PRib-PP (final concentration of 200 mM) was then added to activate the enzyme and the enzyme concentrated by ultrafiltration. The concentration of enzyme was 14 mM and the specific activity 5.9 mmol min − 1 mg − 1. A second sample was prepared by the addition of 1.2 M KCl to the enzyme prepared as above. The concentration of enzyme in this sample was 12 mM and the specific activity after 40 min was zero. The molecular weight of the enzyme in these two samples was determined by sedimentation equilibrium in a Beckman XL-I analytical ultracentrifuge operated at 4°C. Angular velocities of 12 000 and 18 000 rpm were used to generate distributions that were recorded spectrophotometrically at 280 nm and analysed in terms of the sedimentation equilibrium equation for a single solute, namely: A(r) = A(ra)exp[M(1−6r)v 2(r 2 −r 2a)/2RT]

(1)

A(r), the absorbance at radial distance r, is thus related to the corresponding absorbance, A(ra), at the air-liquid meniscus by this exponential expression in terms of solute molecular mass (M), its partial specific volume (6), the buffer density (r),

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the angular velocity (v), the universal gas constant (R) and the absolute temperature (T), as well as the difference between the squares of the two radii. The partial specific volume of HGXPRT was taken as 0.747 ml g − 1, the value calculated from the amino acid composition [21].

3. Results

3.1. Expression of recombinant Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and human hypoxanthine–guanine phosphoribosyltransferase The sequence determined for P. falciparum HGXPRT cDNA was identical to that of Vasanthakumar et al. [5], differing in one position from that reported by King and Melton [22]. It confirms threonine at position 101 of the protein rather than methionine. The levels of expression of P. falciparum HGXPRT and human HGPRT, in terms of activity in cell lysates (measured against guanine; pH 8.5), differed by a factor of 20. For the P. falciparum enzyme, 2 l of cell culture contained 75 units of activity compared with 1500 units for the human enzyme. Both enzymes have been successfully expressed on 20 occasions and the levels of expression are highly reproducible. Based on the specific activities of the purified enzymes (see below; Table 1), each litre of cell culture contained : 8 mg of the Plasmodium enzyme or 15 mg of the human enzyme, showing that similar levels of HGXPRT and HGPRT protein are expressed for both enzymes.

3.2. Purification of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase Two methods were developed to purify P. falciparum HGXPRT. In method (i), the E. coli cell lysate was treated with DNAase and protamine sulfate (as described in Section 2.6) to remove the DNA. P. falciparum HGXPRT in the supernatant from this step was then purified by chromatography on Hg-Sepharose. The protein in the pooled

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Table 1 Specific activities of recombinant Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and human hypoxanthine–guanine phosphoribosyltransferase Enzyme

P. falciparum Humanc

Specific Activity (mmol min−1 mg−1) guanine

hypoxanthine

xanthine

allopurinol

5.4a 1.5c 46a,d 34c,d

1.4a 0.77c 27a 19c

3.0a,b 7.44b,c NAe NAe

3.0 NDe 0.27a NDe

a

0.1 M Tris–HCl, 110 mM MgCl2, pH 8.5. These specific activities were determined in the presence of the following concentrations of xanthine: 543 mM (pH 8.5) and 314 mM (pH 7.4). Because the K app values are 420 and 189 mM, respectively (Table 2), under these conditions, the values quoted here m are lower than the maximum specific activity using xanthine as the purine substrate. c 0.1 M Tris–HCl, 12 mM MgCl2, pH 7.45. d These activities were also determined at 30°C to allow comparison with those reported in the literature [16]. For the human enzyme, the specific activity at pH 7.45 was 47 mmol min−1 mg−1. e NA, no detectable activity. ND, not determined. b

active fraction from this column was found to be homogeneous by SDS-PAGE and to have a specific activity of 4 mmol min − 1 mg − 1 (pH 8.5, guanine as the purine substrate). In method (ii), DEAE-cellulose chromatography was used as the first step. P. falciparum HGXPRT did not bind to the resin under the conditions used. However, the step efficiently removed DNA and a number of contaminating proteins, as shown in Fig. 1. Fractions from the DEAE-cellulose column were completely inactive. Enzyme-containing fractions were

Fig. 1. SDS-polyacrylamide gel electrophoresis (silver stained) of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase. Lane a, molecular weight standard; lane b, crude lysate; lane c, pooled fractions from diethylaminoethyl-cellulose chromatography; and lane d, purified enzyme.

pooled on the basis of SDS-PAGE and further purified on Hg-Sepharose as in method (i). Fig. 1 shows that after Hg-Sepharose chromatography, method (ii) gave a single protein band in the expected mass range on SDS-PAGE. Full activity could be restored to this pooled fraction, as described below. Method (ii) became the preferred method because it reproducibly gave higher yields of protein. From four separate preparations of P. falciparum HGPRT (Method ii), : 10 mg of purified enzyme was obtained each time from 2.5 l of cell culture. Affinity chromatography on GMP-Sepharose was used successfully as a key step in purification of the human enzyme. This step was initially unsuccessful with the P. falciparum enzyme and was not pursued because of the success with HgSepharose. The mass spectrum of purified P. falciparum HGXPRT gives a molecular mass of 26 2299 2 Da, in excellent agreement with the calculated mass of 26 232 Da. For recombinant human HGPRT (C22A, C105A, C205A), the mass spectrum gives a mass of 24 3529 1 Da which also agrees well with the calculated molecular mass of 24 353 Da. These results confirmed that, in both instances, the N-terminal methionine had been cleaved and the new N-terminal amino acid residue had not been acetylated.

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3.3. Stability and acti6ation Using method (i), active P. falciparum HGXPRT (specific activity of 4 mmol min − 1 mg − 1) could be purified to homogeneity in one day. However, maintaining freshly purified enzyme in 0.01 M phosphate, pH 6.8, containing 10 mM DTT and 1 mM PRib-PP at 4°C led to a rapid and almost complete loss of activity e.g. 90% loss of activity in 48 h. Attempts were therefore made to find conditions which would lead to stabilisation of the activity and to reactivation of the inactive P. falciparum HGXPRT purified by method (ii). It was found that the activity of P. falciparum HGXPRT samples purified by method (i) could be maintained for extended periods by storage at 4°C in 0.01 M phosphate, pH 6.8, containing 1 mM DTT, 60 mM Hx, 200 mM PRib-PP. The effects of Hx, PPi, PRib-PP and DTT were further investigated by dialysis into 0.01 M phosphate, pH 7.1, containing (i) 60 mM Hx, 200 mM PPi, 1 mM DTT; (ii) 1 mM DTT alone; or (iii) no additives. The change in activity for samples (i) and (iii) is shown in Fig. 2. In the presence of DTT alone (sample ii), the activity was identical to that in the presence of 60 mM Hx, 200 mM PPi, 1 mM DTT after 96 h. After 3 weeks at 5°C, samples (i) and (ii) retained 1000 units ml − 1 while sample (iii) was completely inactive. If, however, the enzyme was stored in 60 mM Hx, 1 mM DTT and 200 mM PRib-PP, there was no loss of activity over the time period (Fig. 2). In a separate experiment, fully active P. falciparum HGXPRT was dialysed into 0.01 M phosphate, 1 mM DTT, 400 mM PRib-PP for 8 h. This resulted in a loss of 56% of its activity, showing that Hx is required, as well as PRib-PP, to maintain full activity. As shown above, purification by method (ii) resulted in a homogeneous preparation of enzyme which was totally inactive. Building on the results above, the effect of addition of 60 mM Hx and 200 mM PRib-PP to this enzyme (in 0.01 M phosphate, pH 6.8, containing 1 mM DTT) was investigated. Gradual recovery of activity was observed, reaching a maximum value after :72 h. In five such preparations of P. falciparum HGXPRT, the maximum specific ac-

Fig. 2. Time course for the change in activity of Plasmodium falciparum hypoxanthine – guanine – xanthine phosphoribosyltransferase after dialysis into 0.01 M phosphate, pH 7.1 (sample iii, ) or the same buffer containing 60 mM Hx, 200 mM pyrophosphate, 1 mM dithiothreitol (DTT) (sample i, “); undialysed enzyme, stored in 0.01 M phosphate, pH 6.8, 60 mM Hx, 200 mM 5-phospho-a-D-ribosyl-1-pyrophosphate, 1 mM DTT ().

tivities achieved in the reactivation process were 5.4, 5.5, 5.2, 5.5, and 5.9 mmol min − 1 mg − 1. We conclude from these results and the maximum specific activity achieved by method (i) with no activation (4 mmol min − 1 mg − 1) that the specific activity of homogeneous and fully active P. falciparum HGXPRT in the standard assay at pH 8.5 is :5.6 mmol min − 1mg − 1. In analogous experiments, addition of PRib-PP (200 mM) and MgCl2 (10 mM) gave only partial reactivation followed by a gradual loss of activity. After storage for 4–8 weeks at 5°C in 0.01 M phosphate, pH 6.8, 1 mM DTT, 60 mM Hx and 200 mM PRib-PP, the specific activity of the P. falciparum HGXPRT was redetermined, showing only a small loss of activity ( 0 7%) over this time period.

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3.4. Substrate specificities of the two enzymes Specific activities of P. falciparum HGXPRT and human HGPRT towards guanine, Hx, xanthine and allopurinol at pH 8.5 and 7.45 are listed in Table 1. The K app m values for these purine bases are given in Table 2, together with K app m values for PRib-PP with guanine, Hx and xanthine. The ratio) towards each catalytic efficiency (kcat/K app m of these bases at pH 8.5 is also summarized. The efficacy of the purine analogue, allopurinol, as a substrate of P. falciparum HGXPRT and human HGPRT was examined by reversed phase HPLC. Good separation of allopurinol (54 mM) from the products of reaction with the two enzymes in the presence of PRib-PP (952 mM) was obtained (data not shown). Allopurinol was converted to allopurinol ribonucleoside 5%-monophosphate by both enzymes, but these data confirmed that it is a much better substrate for the Plasmodium enzyme.

many weeks without any loss of activity (de Jersey and Keough, unpublished results), showing that this high concentration of salt does not affect the

3.5. Inhibition by mercuric chloride P. falciparum HGXPRT was dialysed into 0.01 M phosphate, 1 mM DTT, pH 7.1 giving a specific activity of 3.0 mmol min − 1 mg − 1 (pH 8.5, guanine as the purine substrate). HgCl2 (3 mM) caused complete inactivation within 3 min at 0°C. Human HGPRT enzyme was dialysed into the same buffer but with 10 mM Mg2 + for 2 h without any loss of activity. This enzyme was also completely inhibited by HgCl2 treatment as above.

3.6. Effect of KCl on the acti6ity and subunit structure of the two enzymes P. falciparum HGXPRT was inactivated by KCl (Fig. 3). PRib-PP alone had no effect on this inactivation but in the presence of Hx, and PRibPP, the rate of inactivation by KCl was slower. Similar experiments with human HGPRT showed no loss of activity in the presence of 1.2 M KCl. The human enzymes, both the recombinant wild type and the mutant form used here, are frequently stored in 0.01 M Tris – HCl, 0.01 M MgCl2, 1.2 M KCl, 200 mM PRib-PP, pH 7.4, for

Fig. 3. Effect of KCl on the activity of Plasmodium falciparum hypoxanthine– guanine – xanthine phosphoribosyltransferase. (A) Enzyme dialysed into 0.01 M phosphate, 1 mM dithiothreitol (DTT), pH 7.1. ( ) no additives; () +5-phospho-a-Dribosyl-1-pyrophosphate (PRib-PP), 1.5 M KCl; (“) −PRib-PP, 1.5 M KCl; () +PRib-PP, 0.74 M KCl; () − PRib-PP, 0.74 M KCl. (B) Enzyme dialysed into 0.01 M phosphate, 1 mM DTT, pH 7.1, 60 mM Hx, 200 mM pyrophosphate. ( ) no additives; (“) +PRib-PP, 1.5 M KCl; () − PRib-PP, 1.5 M KCl; () +PRib-PP, 0.74 M KCl; () −PRib-PP, 0.74 M KCl.

M

)

1.5

0.75

0.005

52 9 5c 0.11

7.5

8.39 0.7c

134 9 15b

2.7

11.891c

1269 13b

3.19 0.9b 3.49 1.0c

NA

NA

NA

NA NA

Xanthine

0.0006

135 9 16b

Allopurinol

b

Measured in the presence of 1 mM 5-phospho-a-D-ribosyl-1-pyrophosphate. Conditions: 0.1 M Tris–HCl, 110 mM MgCl2, pH 8.5. c Conditions: 0.1 M Tris–HCl, 12 mM MgCl2, pH 7.45. d Limited sensitivity of the spectrophotometric assay precluded accurate estimations of these apparent Michaelis constants for the P. falciparum enzyme at pH 7.45. e Measured in the presence of the following concentrations of purine base: guanine, 60 mM; hypoxanthine, 60 mM; xanthine, 205 (pH 8.5); and 336 mM 9 (pH 7.4).

a

(ms

−1 a,b

23 9 4c

−1

40 9 8c

kcat/K app m

168 99b

59.8 9 9.5b

362 9 33b

1.99 0.3b 1.99 0.4c

K app (5-phospho-a-D-ribosyl-1-pyrophosphate) m (mM)e

11.7 9 1.3b

420 912b 189 918c

0.9 90.08b B0.2c,d

1.4 9 0.4b B1c,d

K app (purine) (mM)a m

Hypoxanthine

Guanine

Xanthine

Hypoxanthine

Guanine

Allopurinol

Human HGPRT

P. falciparum HGXPRT

Parameter

Table 2 Comparison of kinetic parameters for Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) and human hypoxanthine–guanine phosphoribosyltransferase (HGPRT)

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strength, an action that also inactivates the enzyme.

4. Discussion

Fig. 4. Sedimentation equilibrium distributions for Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase in 0.01 M phosphate, pH 6.9, containing 60 mM hypoxanthine, 200 mM 5-phospho-a-D-ribosyl-1-pyrophosphate, and 5 mM dithiothreitol (“)(specific activity of 5.9 mmol min − 1 mg − 1); and in the same medium supplemented with 1.2 M KCl () (zero activity).

activity and, therefore, the structure of the active form of this enzyme. Fully active P. falciparum HGXPRT (specific activity of 5.9 mmol min − 1 mg − 1) and the enzyme which had lost activity completely after treatment with KCl were used in sedimentation equilibrium experiments. The sedimentation equilibrium distributions obtained for these two samples at 12 000 rpm and 4°C, shown in Fig. 4, clearly differ markedly in form. Nonlinear regression analysis of the data in terms of Eq. (1) with the buoyant molecular mass, M(1− 6r), and the absorbance at the meniscus, A(ra), as curve-fitting parameters yielded values of 27 200 and 14 400 ( 9 100) Da for the product of molecular mass and the buoyancy correction term in the absence and presence of KCl, respectively. These values have been determined by global curve-fitting of sedimentation equilibrium distributions obtained at 18 000 rpm as well as those at 12 000 rpm shown in Fig. 4 and correspond to values of 107 000 and 59 000 Da for M at low and high ionic strengths. Given the subunit molecular mass of 26 232 Da that is calculated from the amino acid sequence, the enzyme exhibits tetrameric quaternary structure in the absence of KCl — conditions where it is fully active. Essentially complete dissociation of the tetramer into dimeric HGXPRT is effected by elevation of the ionic

Active P. falciparum HGXPRT has been successfully expressed in E. coli cells using the two plasmid system developed earlier for human HGPRT [7]. Both recombinant human HGPRT and P. falciparum HGXPRT have been purified to homogeneity as shown by SDS-PAGE (Fig. 1) and mass spectrometry. In both instances, the N-terminal methionine residue has been cleaved and no other post-translational modification has occurred as indicated by the excellent agreement between measured and calculated molecular masses. For the human enzyme, studies in our laboratory have detected no difference in catalytic properties between recombinant, mutant recombinant, and erythrocyte preparations (de Jersey and Keough, unpublished). Therefore, the lack of Nterminal acetylation has not affected the catalytic efficiency of the human enzyme. Since the Plasmodium enzyme has never been isolated from the parasite, it is unknown whether the naturally occurring enzyme is acetylated or has any other post-translational modifications. A major problem in studying HGPRTs and HGXPRTs from various sources has been their instability during purification and storage. The addition of DTT and PRib-PP has been successful in stabilizing several HGPRTs including human. HGPRT from the avian malaria parasite, P. lophurae, lost activity very rapidly during the purification procedure [23] and the addition of PRib-PP to the column buffers did not stabilise it. This instability also posed a serious problem in previous attempts to purify and characterize P. falciparum HGXPRT as mentioned in the introduction. The addition of 60 mM Hx to enzyme solutions in 0.01 M phosphate, pH 6.8 or 7.1, containing 200 mM PRib-PP and 1 mM DTT was found to be totally effective in maintaining full activity for periods of at least 2 months at 4°C. Enzyme samples which had lost activity during purification by method (ii) could be returned to full activity by the addi-

D.T. Keough et al. / Molecular and Biochemical Parasitology 98 (1999) 29–41

tion of 60 mM Hx and 200 mM PRib-PP to the buffer. As a result of the work described above, P. falciparum HGXPRT is available for the first time in pure and stable form, in quantities sufficient for structural as well as functional characterization. These stability and reactivation experiments suggest that PRib-PP is able to bind to the Plasmodium enzyme in the presence of Hx and absence of Mg2 + . In contrast, human HGPRT only binds PRib-PP in the presence of Mg2 + [16], this interaction with Mg2 + PRib-PP complex being a prerequisite for binding of the purine base [16]. One obvious functional difference between the Plasmodium and human enzymes is in their catalytic efficiency in the standard assay. The specific activity of the human enzyme (using either guanine or Hx as the purine base) is 15 – 40-fold greater than that of the P. falciparum enzyme, when they are assayed under the same conditions (Table 1). Similar low specific activities have been reported for other parasite HGXPRTs [24,25]. Given the central role these enzymes are thought to play in parasite metabolism, it may be speculated that either the assay conditions do not reflect those in vivo or that, in the parasite, the enzymes are present at much higher concentrations than in human cells. In support of the latter proposition, the concentration of HGPRT in the cells of Leishmania dono6ani has been reported to be 2 –70-fold higher that in human cells [18]. A second major difference between the P. falciparum and human enzymes, in terms of usage of the naturally occurring purine base substrates, is the ability of P. falciparum HGXPRT to utilize xanthine. The specific activity using xanthine is similar at pH 8.5 to that using guanine, but it is five times greater when measured at pH 7.4 (Table 1). The specific activity for Hx is lower than for guanine and xanthine. Human HGPRT has similar specific activities with guanine and Hx and no activity could be detected with xanthine at either pH value (Table 1). Relative specific activities for the 6-oxopurine bases (Table 1) and the K app m data of Table 2 are consistent with the results of Queen et al. [4] obtained for partially purified P. falciparum enzyme.

39

The purine analogue, allopurinol, has been widely used as a xanthine oxidase inhibitor in the treatment of gout and has no known deleterious effects on mammalian cells. Allopurinol has been reported to inhibit the growth of Leishmania brazi, dono6ani and mexicana in culture [26] and exhibited chemotherapeutic effects in preliminary clinical trials against leishmaniasis and Chagas disease [27]. This purine base analogue has proved to be a good substrate for P. falciparum HGXPRT, giving a specific activity comparable to those for guanine and Hx (Table 1). For Trypanosoma brucei HGPRT, the specific activity towards allopurinol was reported to be : 8% of that for guanine [28]. Allopurinol is a very poor substrate for human HGPRT, with a specific activity of only 0.6% of that found for guanine of 135 mM (Table 2). Using (Table 1) and a K app m the ratio kcat/Km as a measure of specificity, allopurinol is :200 times better as a substrate for P. falciparum HGXPRT than for human HGPRT. By the same measure, Hx is 3.7 times better as a substrate for human HPRT. Mercuric ion rapidly and completely inactivated both Plasmodium and human HGPRT, though the molecular explanation for the inactivation is not clear. In the human enzyme, none of the four cys residues present per subunit is necessary for catalysis [11], but it remains to be seen if any of the cysteine residues are essential for the Plasmodium enzyme. Both the human and Plasmodium enzymes lose activity in absence of DTT (Section 3.3; [11]). For the human enzyme, this has been shown to be due to the formation of inter- and intra-subunit disulfide bonds [11]. Another significant difference between the human and parasite enzymes is the effect of salt. KCl rapidly inactivated the Plasmodium enzyme (Fig. 3) but had essentially no effect on the human enzyme. The effect of KCl may explain the loss of activity which occurred during ion-exchange chromatography of P. falciparum HGXPRT [4]. Using ultracentrifugation (Fig. 4), it has been shown that the fully active enzyme is tetrameric and that addition of KCl results in dissociation of tetramers to dimers. Based on gel filtration data, Queen et al [4] suggested that the enzyme exists as a dimer. However, those experi-

40

D.T. Keough et al. / Molecular and Biochemical Parasitology 98 (1999) 29–41

ments were performed in 0.15 M phosphate, pH 6.5, containing 0.1 M NaCl, and in the light of the ultracentrifugation studies presented here, it is likely that dissociation to dimers had occurred on the column. In conclusion, P. falciparum HGXPRT has been purified to homogeneity in sufficient quantities to allow detailed kinetic and structural studies. The data presented here show significant differences between the human and parasite enzymes, indicating that parasite-specific inhibitors are feasible. Our current studies are directed towards determining the three dimensional structure of the parasite enzyme and developing specific inhibitors.

[8]

[9]

[10]

[11]

[12]

Acknowledgements We would like to acknowledge the support of grants from the Australian Research Council and to thank Michael Jacobsen for assistance in the ultracentrifugation studies and Greg Dulley for the initial HPLC analyses using allopurinol and hypoxanthine as substrates.

[13]

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