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Interaction of human 3-phosphoglycerate kinase with l -ADP, the mirror image of d -ADP
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Biochemical and Biophysical Research Communications 366 (2008) 994–1000 www.elsevier.com/locate/ybbrc
Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP Andrea Varga a, Judit Szabo´ a, Bea´ta Flachner a, Be´atrice Roy b, Peter Konarev Dmitri Svergun c,d, Pe´ter Za´vodszky a, Christian Pe´rigaud b, Tom Barman e, Corinne Lionne e, Ma´ria Vas a,*
c,d
,
a
e
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina Str. 29, P.O. Box 7, H-1518 Budapest, Hungary b Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247 CNRS, Universite´s Montpellier 1 et 2, case courrier 1705, Universite´ Montpellier 2, Place Euge`ne Bataillon, 34095 Montpellier cedex 5, France c European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany d Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia Centre d’e´tudes d’agents Pathoge`nes et Biotechnologies pour la Sante´ (CPBS), UMR 5236 CNRS, Universite´s Montpellier 1 et 2, Institut de Biologie, 4 bd Henri IV, CS69033, 34965 Montpellier cedex 2, France Received 30 November 2007 Available online 26 December 2007
Abstract L-Nucleoside-analogues, mirror images of the natural D-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound L-MgADP with hPGK. L-MgADP is a good substrate with kcat and Km values of 685 s1 and 0.27 mM, respectively. Double inhibition studies suggest that L-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the D- and L-enantiomers (e.g. the effect of Mg2+), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hingebending domain closure, as indicated by SAXS measurements. Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Human 3-phosphoglycerate kinase; Enantioselectivity; Nucleotide substrates; Enzyme kinetic analysis; Microcalorimetry; Small angle X-ray scattering
It has been shown that the glycolytic enzyme, 3-phosphoglycerate kinase (PGK) may play an important role Abbreviations: AMP-PCP, b,c-methylene-adenosine-5 0 triphosphate; AMP-PNP, b,c-imido-adenosine-5 0 triphosphate; 1,3-bPG, 1,3-bisphosphoglycerate; Pi, inorganic phosphate; DSC, differential scanning calorimetry; DTT, dithiothreitol; GAP, D-glyceraldehyde-3-phosphate; GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); HIV, human immunodeficiency virus; Nbs2, Ellman’s reagent, 5,5 0 -dithiobis-(2-nitrobenzoic acid); 3-PG, 3-phosphoglycerate; PGK, 3-phospho-D-glycerate kinase or ATP, 3-phospho-D-glycerate 1-phosphotransferase (EC 2.7.2.3); hPGK, human PGK. * Corresponding author. Fax: +36 1 466 5465. E-mail address: [email protected] (M. Vas). 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.061
in the activation of some antiviral L-nucleoside analogue prodrugs [1,2]. The unnatural L-configuration of the nucleosides are favoured in anti-HIV therapy compared to their natural D-counterparts [3]. The drugs are administrated to the patient in the form of nucleoside and require sequential steps of phosphorylation to reach their active triphosphate form. Among other kinases, PGK seems to be the most effective one in phosphorylation of compounds with L-configuration [2]. Here the question arises about the structural basis of phosphorylation of Lnucleoside diphosphates by PGK. With natural substrates extensive structural–functional studies have been carried out on PGK (e.g. [4–9]). This accumulated
A. Varga et al. / Biochemical and Biophysical Research Communications 366 (2008) 994–1000
knowledge helps in understanding the structural basis of phosphorylation of L-nucleotides and in developing new compounds with antiviral effect. In fact, there is a continuous demand to develop new drugs because of the resistance of HIV reverse transcriptase due to its high mutation rate [10]. Here we studied the pharmacologically important phosphorylation step as catalysed by hPGK in the forward reaction of glycolysis. As a first attempt, we carried out comparative kinetic, binding and inhibitory studies of hPGK using both L- and D-ADP as substrates or inhibitors. These studies were also completed by microcalorimetry and SAXS. The experiments revealed that L-ADP is almost as a good substrate of hPGK as its natural enantiomer. The binding interactions of L-MgADP highly resemble those with D-MgADP, but there are well-defined differences, e.g. Mg2+ seems to be less effective in strengthening the binding of L-ADP. Materials and methods Protein and reagents. Expression and purification of recombinant hPGK was described earlier [4,11]. The protein solution (20 mg/ml, in a buffer 50 mM Tris, pH 7.5, containing 1 mM EDTA and 1 mM DTT) was stored at 80 °C. Protein concentration was determined using an extinction coefficient of 27,900 M1 cm1 at 280 nm. GAPDH was prepared from pig muscle and stored as a microcrystalline suspension [12]. Crystals of this auxiliary enzyme were dialysed against the buffer given above. GAPDH concentration was calculated using 144,000 M1 cm1 at 280 nm for the tetramer. 3-PG and GAP were from Boehringer Mannheim, and D-ADP, D-ATP, NAD, NADH and adenosine from Sigma–Aldrich. 1,3-bPG was prepared from GAP according to Negelein [13] with the modification described by Furfine and Velick [14]. Ellman’s reagent (Nbs2) was obtained from Serva. LADP was synthesised starting from peracetylated L-ribose as described by He et al. [15]. The Mg2+ complexes of the nucleotides were obtained by the addition of MgCl2 knowing the Kd values of the Mg complexes of ADP (0.6 mM) or ATP (0.1 mM) [16,17]. Steady state kinetic and inhibitory studies. The activity of PGK was measured with either D- or L-MgADP and 1,3-bPG as substrates at 20 °C, in the above buffer. Prior to initiating the reaction by PGK, the substrate 1,3-bPG was formed in the activity assay mixture from 1 mM GAP, 0.5 mM NAD and 50 mM Na-phosphate under the action of GAPDH. The formation of 1,3-bPG was indicated by the spectral signal of NADH (e366 = 3300 M1 cm1) formed simultaneously. A large molar excess of GAPDH compared to PGK assures the faster production of 1,3-bPG (compared to its consumption by PGK, since the turnover rates of PGK (1085 s1) and GAPDH (1040 s1) are similar. Upon starting the PGK reaction, further NADH production was followed at 366 nm, equivalent to the amount of 1,3-bPG consumed by PGK [18]. As Pi is a competitive inhibitor of PGK (with respect to ADP) and as its concentration used was high (50 mM), the obtained Km values (Kmapp) were corrected by Eq. (1):
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consumption of NADH was followed spectrophotometrically at 340 nm (e340 = 6220 M1 cm1) [19]. Double inhibition experiments [20] in the presence of the two inhibitors (MgADP plus adenosine) were also carried out. Thiol reactivity studies. Kd values of ligand binding to PGK was determined on the basis of the protective effects of ligands against modification of the fast-reacting thiol groups. The rate of thiol modification by Nbs2 was followed at 412 nm (e412 = 14,150 M1 cm1) and analysed as described by Kova´ri et al. [8]. DSC experiments. The measurements were carried out in a MicroCal VP-DSC-type microcalorimeter (MicroCal Inc.) with a cell volume of 0.51 mL, at a constant scan rate of 60 °C/h. Tm values were determined in the presence of various concentrations of either D- or L-ADP. The data were analysed using MicroCal Origin 5.0. The maximum effects of ligands on the Tm values were determined by extrapolating the observed changes to infinite nucleotide concentrations using Eq. (2): T measured ¼ T min þ
ðT max T min Þ ½N K þ ½N
ð2Þ
where Tmin is measured in the absence of the nucleotide ligand, N, and Tmax is the extrapolated value at infinite [N]. SAXS measurements and data processing. Synchrotron radiation Xray scattering data were collected on the X33 beam line at the Hamburg EMBL Outstation [21] as described in Varga et al. [22]. Solution of hPGK (5–15 mg/mL, i.e. about 0.1–0.3 mM in the storage buffer), its binary complexes with 1,3-bPG (0.9 mM), D- or L-MgADP (2.2 mM), and the ternary complexes with 3-PG (5.7 mM) plus MgADP (2.2 mM) as well as with 1,3-bPG (0.9 mM) plus MgADP (2.2 mM for both enantiomers) were measured using a MAR345 Image Plate at a sample˚ , covering the detector distance of 2.4 m and wavelength of 1.5 A ˚ 1 (s = 4p sin (h)/k where momentum transfer range 0.012 < s < 0.45 A 2h is the scattering angle). The data were processed by PRIMUS [23] and the scattering patterns from the atomic models of the X-ray coordinates were computed using CRYSOL [24].
Results and discussion Characterisation of L-ADP as substrate or inhibitor: steady state kinetics
ð1Þ
ATP formation from 1,3-bPG and D- or L-MgADP PGK catalyses the reversible phospho-transfer between 1,3-bisphosphoglycerate (1,3-bPG) and MgADP: 1,3bPG + MgADP M 3-PG + MgATP. Usually the reaction is studied in the backward direction of glycolysis due to the sensitivity of 1,3-bPG towards hydrolysis. Here we followed the forward reaction using 1,3-bPG produced directly in the assay mixture. Fig. 1A shows the saturation curves with D- or L-MgADP. Thus, L-MgADP is almost as a good substrate for hPGK as the natural D-MgADP. The kcat and Km values with L and D enantiomers are 685 and 1085 s1, and 0.27 and 0.12 mM, respectively. The Km value for D-MgADP agrees well with that obtained earlier using the HK-G6PDH coupled assay [11].
where KI is the inhibition constant for Pi. KI value was obtained by a method involving HK and G6PDH [11] and was estimated to be 17.7 mM. Under the conditions used, GAP and NAD did not inhibit the PGK. The inhibitory properties of D- and L-MgADP were tested using 3-PG and MgATP as substrates. The reaction is again coupled to GAPDH reaction by reducing the product 1,3-bPG in the presence of NADH. The
D- and L-MgADP as inhibitors with MgATP and 3-PG as substrates In the PGK-catalysed reaction starting from 3-PG and MgATP, the product D-MgADP was shown to be a competitive inhibitor with respect to MgATP [25]. Fig. 1B compares the inhibitory effects of D- and
Km ¼
K app m KI ½Pi þ K I
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Fig. 1. Comparison of the kinetics of hPGK using L- and D-ADP either as substrate (A) or inhibitor (B–D). In (A) activities of 4 nM hPGK were recorded as a function of the L- (j) or D-ADP (d) concentration, in the presence of 15 mM MgCl2, 1 mM GAP, 0.5 mM NAD, 50 mM inorganic phosphate and the appropriate excess of GAPDH. The curves were fitted by the Michaelis–Menten equation. In (B–D) the PGK activity was monitored using MgATP and 3PG as substrates. The experiments were carried out with 8.8 nM (B) or 26.6 nM (C,D) hPGK in the presence of 10 mM (B) or 0.5 mM (C,D) 3-PG. All mixtures contained 0.5 mM ATP and 12.5 mM MgCl2. Activities were determined as a function of the L- (j, h) or D-MgADP (d, s) concentration. In (C,D) the inhibition was measured both in the absence (j, d) and in the presence of 20 mM adenosine (h, s) and plotted according to the model of Yonetani and Theorell [20].
L-MgADP
on this reaction. The Ki values for D- and L-MgADP are 35 and 63 lM, respectively. Thus, the relative values of the Ki resemble those of the Km values and similarly indicate a slight weakening of the interactions of L-MgADP with hPGK compared to those of D-MgADP. Double inhibition experiments To explore whether both D- and L-MgADP compete for the same adenosine-binding pocket that is located in the Cdomain of PGK, double inhibition experiments, similar to those published earlier with D-MgADP [9], were carried out in the presence of either D- or L-MgADP and adenosine. The Yonetani–Theorell plots [20] in Fig. 1C and D show parallel lines, indicating that the inhibitory effect of either D- or L-MgADP and adenosine are exclusive. Thus, it appears that D- and L-MgADP compete for the same adenosine site of PGK.
Effect of Mg2+ on the interactions of L- and D-ADP with hPGK: equilibrium studies For D-ADP binding the strengthening effect of Mg2+ is well documented [25,26]. Comparison of the binding constants of Mg2+-free- and MgADP showed an interesting difference between the two enantiomers, as revealed by thiol-reactivity studies (cf. below). The interaction of 2+ L-ADP with hPGK is much less strengthened by Mg (6-fold decrease in Kd, cf. Table 1) than that of D-ADP (at least 20-fold decrease in Kd [25]). The high-resolution crystal structure provides an explanation for the strengthening effect of Mg2+: the metal ion interacts with both the a- and b-phosphates of ADP as well as with the conserved Asp 374 located in helix 13 between the two domains [5]. Thus, the above differences may indicate different types of interactions of D- and L-ADP with hPGK through Mg2+.
A. Varga et al. / Biochemical and Biophysical Research Communications 366 (2008) 994–1000 Table 1 Dissociation constants (Kd) for the D- and L-enantiomers of ADP with or without Mg2+ Enantiomeric form
Ligand
Kd (lM)
D
ADP MgADP
590 ± 200 24 ± 2
L
ADP MgADP
410 ± 130 71 ± 18
The Kd values were determined using the method thiol reactivity, described in Materials and methods.
Effect of D- and L-ADP on the PGK conformation: biophysical studies Protective effect of D- and L-ADP against modification of the reactive thiols of PGK In a previous study, we showed that the reactivity of the two thiol groups located in helix 13 are sensitive to the presence of the substrates of PGK, in particular MgADP. Furthermore, MgADP has a much larger protective effect against thiol modification than Mg2+-free ADP [9], in accordance with the crystallographic data, showing ligation of the phosphates of ADP through Mg2+ to Asp 374 in helix 13 [5]. This interaction with MgADP makes helix 13 highly ordered as reflected by the low crystallographic average B-factor values for the main chain atoms of helix 13 [5,8]. Since ordering of helix 13 results in the shielding of the two reactive thiols and reduces their reactivity [8], testing of thiol reactivity is a sensitive tool for detection of substrate-caused local conformational changes. Here, the effects of L-ADP and L-MgADP were tested in the same way. The rate constants of thiol modification in the absence and presence of saturating amounts of D- or L-MgADP were found to be 1800 ± 22 and 30 ± 10 M1 s1, respectively (data not illustrated). This suggests that the binding modes of the phosphates of the MgADP enantiomers are approximately the same relative to the position of helix 13 and that both ligands cause similar local conformational changes around the reactive thiols. From dependences of the rate constants of thiol modification on the concentration of the nucleotides, their Kd values were also obtained and are given in Table 1. From the values we conclude that in spite of their similar effects on the conformation of helix 13, the interaction with Asp 374 (through Mg2+) is possibly weaker in case of L-MgADP. Protective effect of the nucleotides against unfolding Previous DSC studies showed that the substrates protect PGK against heat denaturation [9,27]. Here again, MgADP exerted much larger protection than Mg-free ADP (Fig. 2). From the experiments at different concentrations of L-MgADP, we could extrapolate to Tm at infinite concentration of the nucleotide. The extrapolated Tm value (59.5 ± 0.1 °C) is very similar to that obtained with D-MgADP. Thus, in spite of its mirror image conforma-
997
tion, by this method L-MgADP affects the overall conformation of PGK similarly to D-MgADP. Substrate-induced domain closure as detected by SAXS PGK is a monomer protein that contains two globular domains. The N-terminal domain binds 1,3-bPG or 3-PG and the C-terminal one contains the nucleotide-binding site. In the apoenzyme, the substrate binding sites are too far apart to allow phospho-transfer but upon the binding of both substrates an extensive ‘‘hinge binding motion’’ occurs that leads to the approximation of the sites [6,7,22]. In the latter work we have demonstrated that the scattering curves of substrate-free hPGK and its various binary and ternary enzyme-substrate complexes in solution correlate well with the theoretical curves calculated from the respective crystal structures, supporting the occurrence of domain closure in the ternary complexes [22]. Thus, the presence of both substrates is required for PGK domain closure. To determine whether or not L-MgADP induces domain closure, we carried out SAXS measurements on the binary EÆL-MgADP and the ternary EÆL-MgADPÆ1,3bPG and EÆL-MgADPÆ3-PG complexes. Thus, in addition to the latter non-functional ternary complex (investigated previously with D-MgADP) here we also studied the functioning L-MgADPÆ1,3-bPG.1 Control measurements were also carried out on the substrate-free PGK and on its binary complex with 1,3-bPG. The importance of the latter SAXS measurements is underlined by the fact that no crystal structure of PGKÆ1,3-bPG is available. The scattering curves obtained are shown in Fig. 3. The deviation of the experimental curves (dots with error bars) from the theoretical ones (continuous lines calculated from various open and closed crystallographic models) is expressed numerically by the discrepancy (v2) values (Table 2). The data show that the substrate-free enzyme exhibits a completely open structure. The binary complexes with LMgADP and D-MgADP gave the best agreement with the open crystal structure of the binary complex with MgATP [9]. The binary complex with 1,3-bPG agrees better with the partially closed conformation observed in the crystal structure of the binary complex with 3-PG [4]. The scattering curves of both functioning and non-functioning ternary complexes including L-MgADP (Fig. 3A) correlate well with the ones computed from the closed crystal structures, Thermotoga maritima [6] and Trypanosoma brucei [7]
1
Although the chemical equilibrium of either D- or L-MgADP phosphorylation reaction favours the formation of MgATP and 3-PG, but due to the high enzyme concentration (0.1–0.3 mM) applied and the extremely strong binding of the small molar excess (0.9 mM) of 1,3-bPG, the value of Kint, i.e. the equilibrium between the bound substrates and products possibly greatly differs from the above equilibrium [28,29] and the amounts of bound L-MgADP and.1,3-bPG are not negligible during the experiment. At any case, the data obtained starting from the complex with L-MgADP and 1,3-bPG, are characteristic of the average of the functioning ternary complexes, containing bound L -nucleotides (L-MgADP.1,3-bPG and L-MgATP.3-PG).
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Fig. 2. Effect of L- and D-MgADP on the thermal stability of hPGK. DSC transition curves (A) were determined in the absence (black dashed line) or in the presence of D-ADP (grey dashed line), D-MgADP (grey solid line) or L-MgADP (black solid line). The melting temperatures were plotted against the L(h) or D-MgADP (s) concentration (B). The free Mg2+ concentration was 2 mM. The solid lines represent the best fits of the experimental data given by Eq. (2).
Fig. 3. Experimental and calculated SAXS scattering curves for PGK obtained with L-MgADP (A) and D-MgADP (B). The experiments were carried out in the absence of substrates (1), or in the presence of 2.2 mM ADP plus 12.5 mM MgCl2 (2), 0.9 mM 1,3-bPG (3), 2.2 mM ADP, 12.5 mM MgCl2 plus 0.9 mM 1,3-bPG (4) or 2.2 mM ADP, 12.5 mM MgCl2 plus 5.7 mM 3-PG (5).
PGKs. The equivalent non-functioning ternary complex with D-MgADP and 3-PG was investigated previously and was found to be between the crystal structures of the binary complex with 3-PG (v2 = 2.049) and of the much better closed Thermotoga maritima PGK ternary complex (v2 = 2.018)) [22]. This marginal difference is, however, at the accuracy limit of the data. The present data provided similar results, not only for the non-functioning . D-MgADP 3PG, but also for the functioning . D-MgADP 1,3-bPG ternary complex (Fig. 3B and Table
2). Thus, we can conclude that, as expected, the ternary complexes, containing D-MgADP, possess largely closed conformations. Taken together, the unnatural L-MgADP in its functional ternary complex induces domain closure of hPGK, in agreement with the present kinetic results. In summary, hPGK can accommodate the mirror image L-enantiomer of MgADP into its nucleotide-binding site and can phosphorylate it, almost as effectively as the natural D-enantiomer. The fine structural details of the interactions of the two substrates (e.g. the interaction with Mg2+)
A. Varga et al. / Biochemical and Biophysical Research Communications 366 (2008) 994–1000
999
Table 2 Comparison of SAXS experimental parameters with those of the crystallographic models SAXS experiments
0
Discrepancy v2 between the scattering from crystallographic models and experimental dataa
Investigated hPGK complexes
) Rg (experimental) (A
Open crystal structures
GNOM method
Guinier method
Pig PGK (no ligand)
No ligand
23.9 ± 0.5
24.2 ± 0.1
1.91
3.72
2.43
2.07
6.02
6.64
Binary 1,3-bPG L-MgADP D-MgADP
23.7 ± 0.6 23.9 ± 0.6 23.7 ± 0.7
24.0 ± 0.2 24.1 ± 0.2 23.8 ± 0.2
1.71 2.38 1.50
2.59 2.80 1.51
2.00 1.70 1.48
1.58 1.93 1.62
3.32 3.02 1.91
3.64 3.71 2.23
22.7 ± 0.5 22.5 ± 0.5 23.6 ± 0.6 23.8 ± 0.5
22.9 ± 0.2 22.7 ± 0.2 23.9 ± 0.2 24.1 ± 0.2
1.58 1.91 1.76 1.43
1.70 1.88 1.83 1.61
1.58 1.82 1.54 1.38
1.55 1.81 1.52 1.35
1.59 1.80 1.58 1.39
1.42 1.73 1.69 1.79
24.25 43.7
24.34 43.2
24.02 43.6
23.97 43.8
23.26 43.7
22.64 45.3
Bs PGK MgADP binary
Pig PGK MgATP binary
Partially closed
Closed crystal structures
Pig PGK 3-PG binary
Tm PGK ternary1c
Tb PGK ternary2c
Ternary L-MgADPÆ1,3-bPG L-MgADPÆ3-PG D-MgADPÆ1,3-bPG D-MgADPÆ3-PG 0
) Rg (theoretical)b (A Molecular massb (kDa) a b c
The minimum values of discrepancy (in bold) indicate the best correlation between SAXS data and crystallographic model. Radius of gyration and molecular mass of the high-resolution models as retrieved from the PDB. Ternary1 and ternary2 denote MgAMP-PNPÆ3-PG and MgADPÆ3-PG ternary complexes, respectively.
in the enzyme active site are different for the D- and L-enantiomers, but these differences do not prevent the occurrence of the catalytic cycle. Based on our results, hPGK can be considered as a promising candidate for assisting in vivo phosphorylation of the pharmacologically potent L-nucleoside analogues with antiviral effects. Acknowledgments The paper was prepared within the framework of the Hungarian-French Intergovernmental Scientific and Technological Cupertino Program of the Hungarian Foundation of Research and Innovative Technology (OMFB00493/2007) as well as by Egide (Balaton program No. 14100ZF). The financial support by OTKA NI 61915 from the Hungarian National Research Foundation as well as the travelling grants for B.F., A.V. and Sz. J. by European Community-Research Infrastructure Action under the FP6 ‘‘Structuring the European Research Area Programme’’ (No. RII3-CT-2004-506008) are also gratefully acknowledged. D.S. and P.K. acknowledge support from the EU Design Study ‘‘SAXIER’’, contract no. 011934. A.V. was supported by a short term FEBS fellowship. References [1] P. Krishnan, Q. Fu, W. Lam, J.Y. Liou, G. Dutschman, Y.C. Cheng, Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of L-nucleoside analog diphosphates by 3phosphoglycerate kinase, J. Biol. Chem. 277 (2002) 5453–5459. [2] S. Gallois-Montbrun, A. Faraj, E. Seclaman, J.P. Sommadossi, D. Deville-Bonne, M. Ve´ron, Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs, Biochem. Pharmacol. 68 (2004) 1749–1756. [3] C. Mathe´, G. Gosselin, L-nucleoside enantiomers as antivirals drugs: a mini-review, Antiviral Res. 71 (2006) 276–281.
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network as a means of domain closure in 3-phosphoglycerate kinase, FEBS Lett. 580 (2006) 2698–2706. P.V. Konarev, V.V. Volkov, M.V. Petoukhov, D.I. Svergun, ATSAS 2.1, a program package for small-angle scattering data analysis, J. Appl. Cryst. 39 (2006) 277–286. D.I. Svergun, C. Barberato, M.H.J. Koch, CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates, J. Appl. Crystallogr. 28 (1995) 768–773. M. Molna´r, M. Vas, Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase. Correlation between equilibrium dialysis binding and enzyme kinetic data, Biochem. J. 293 (1993) 595–599. A. Merli, A.N. Szila´gyi, B. Flachner, G.L. Rossi, M. Vas, Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: relationship between substrate antagonism and interdomain communication, Biochemistry 41 (2002) 111–119. A. Varga, B. Flachner, E. Gra´czer, S. Osva´th, A.N. Szila´gyi, M. Vas, Correlation between conformational stability of the ternary enzymesubstrate complex and domain closure of 3-phosphoglycerate kinase, FEBS J. 272 (2005) 1867–1885. B.D. Nageswara-Rao, M. Cohn, R.K. Scopes, 31P NMR study of bound reactants and products of yeast 3-phosphoglycerate kinase at equilibrium and the effect of sulfate ion, J. Biol. Chem. 253 (1978) 8056–8060. P.P. Schmidt, F. Travers, T. Barman, Transient and equilibrium kinetic studies on yeast 3-phosphoglycerate kinase. Evidence that an intermediate containing 1,3-bisphosphoglycerate accumulates in the steady state, Biochemistry 34 (1995) 824–832.
Biochemical and Biophysical Research Communications 366 (2008) 994–1000 www.elsevier.com/locate/ybbrc
Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP Andrea Varga a, Judit Szabo´ a, Bea´ta Flachner a, Be´atrice Roy b, Peter Konarev Dmitri Svergun c,d, Pe´ter Za´vodszky a, Christian Pe´rigaud b, Tom Barman e, Corinne Lionne e, Ma´ria Vas a,*
c,d
,
a
e
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina Str. 29, P.O. Box 7, H-1518 Budapest, Hungary b Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247 CNRS, Universite´s Montpellier 1 et 2, case courrier 1705, Universite´ Montpellier 2, Place Euge`ne Bataillon, 34095 Montpellier cedex 5, France c European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany d Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia Centre d’e´tudes d’agents Pathoge`nes et Biotechnologies pour la Sante´ (CPBS), UMR 5236 CNRS, Universite´s Montpellier 1 et 2, Institut de Biologie, 4 bd Henri IV, CS69033, 34965 Montpellier cedex 2, France Received 30 November 2007 Available online 26 December 2007
Abstract L-Nucleoside-analogues, mirror images of the natural D-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound L-MgADP with hPGK. L-MgADP is a good substrate with kcat and Km values of 685 s1 and 0.27 mM, respectively. Double inhibition studies suggest that L-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the D- and L-enantiomers (e.g. the effect of Mg2+), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hingebending domain closure, as indicated by SAXS measurements. Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Human 3-phosphoglycerate kinase; Enantioselectivity; Nucleotide substrates; Enzyme kinetic analysis; Microcalorimetry; Small angle X-ray scattering
It has been shown that the glycolytic enzyme, 3-phosphoglycerate kinase (PGK) may play an important role Abbreviations: AMP-PCP, b,c-methylene-adenosine-5 0 triphosphate; AMP-PNP, b,c-imido-adenosine-5 0 triphosphate; 1,3-bPG, 1,3-bisphosphoglycerate; Pi, inorganic phosphate; DSC, differential scanning calorimetry; DTT, dithiothreitol; GAP, D-glyceraldehyde-3-phosphate; GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); HIV, human immunodeficiency virus; Nbs2, Ellman’s reagent, 5,5 0 -dithiobis-(2-nitrobenzoic acid); 3-PG, 3-phosphoglycerate; PGK, 3-phospho-D-glycerate kinase or ATP, 3-phospho-D-glycerate 1-phosphotransferase (EC 2.7.2.3); hPGK, human PGK. * Corresponding author. Fax: +36 1 466 5465. E-mail address: [email protected] (M. Vas). 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.061
in the activation of some antiviral L-nucleoside analogue prodrugs [1,2]. The unnatural L-configuration of the nucleosides are favoured in anti-HIV therapy compared to their natural D-counterparts [3]. The drugs are administrated to the patient in the form of nucleoside and require sequential steps of phosphorylation to reach their active triphosphate form. Among other kinases, PGK seems to be the most effective one in phosphorylation of compounds with L-configuration [2]. Here the question arises about the structural basis of phosphorylation of Lnucleoside diphosphates by PGK. With natural substrates extensive structural–functional studies have been carried out on PGK (e.g. [4–9]). This accumulated
A. Varga et al. / Biochemical and Biophysical Research Communications 366 (2008) 994–1000
knowledge helps in understanding the structural basis of phosphorylation of L-nucleotides and in developing new compounds with antiviral effect. In fact, there is a continuous demand to develop new drugs because of the resistance of HIV reverse transcriptase due to its high mutation rate [10]. Here we studied the pharmacologically important phosphorylation step as catalysed by hPGK in the forward reaction of glycolysis. As a first attempt, we carried out comparative kinetic, binding and inhibitory studies of hPGK using both L- and D-ADP as substrates or inhibitors. These studies were also completed by microcalorimetry and SAXS. The experiments revealed that L-ADP is almost as a good substrate of hPGK as its natural enantiomer. The binding interactions of L-MgADP highly resemble those with D-MgADP, but there are well-defined differences, e.g. Mg2+ seems to be less effective in strengthening the binding of L-ADP. Materials and methods Protein and reagents. Expression and purification of recombinant hPGK was described earlier [4,11]. The protein solution (20 mg/ml, in a buffer 50 mM Tris, pH 7.5, containing 1 mM EDTA and 1 mM DTT) was stored at 80 °C. Protein concentration was determined using an extinction coefficient of 27,900 M1 cm1 at 280 nm. GAPDH was prepared from pig muscle and stored as a microcrystalline suspension [12]. Crystals of this auxiliary enzyme were dialysed against the buffer given above. GAPDH concentration was calculated using 144,000 M1 cm1 at 280 nm for the tetramer. 3-PG and GAP were from Boehringer Mannheim, and D-ADP, D-ATP, NAD, NADH and adenosine from Sigma–Aldrich. 1,3-bPG was prepared from GAP according to Negelein [13] with the modification described by Furfine and Velick [14]. Ellman’s reagent (Nbs2) was obtained from Serva. LADP was synthesised starting from peracetylated L-ribose as described by He et al. [15]. The Mg2+ complexes of the nucleotides were obtained by the addition of MgCl2 knowing the Kd values of the Mg complexes of ADP (0.6 mM) or ATP (0.1 mM) [16,17]. Steady state kinetic and inhibitory studies. The activity of PGK was measured with either D- or L-MgADP and 1,3-bPG as substrates at 20 °C, in the above buffer. Prior to initiating the reaction by PGK, the substrate 1,3-bPG was formed in the activity assay mixture from 1 mM GAP, 0.5 mM NAD and 50 mM Na-phosphate under the action of GAPDH. The formation of 1,3-bPG was indicated by the spectral signal of NADH (e366 = 3300 M1 cm1) formed simultaneously. A large molar excess of GAPDH compared to PGK assures the faster production of 1,3-bPG (compared to its consumption by PGK, since the turnover rates of PGK (1085 s1) and GAPDH (1040 s1) are similar. Upon starting the PGK reaction, further NADH production was followed at 366 nm, equivalent to the amount of 1,3-bPG consumed by PGK [18]. As Pi is a competitive inhibitor of PGK (with respect to ADP) and as its concentration used was high (50 mM), the obtained Km values (Kmapp) were corrected by Eq. (1):
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consumption of NADH was followed spectrophotometrically at 340 nm (e340 = 6220 M1 cm1) [19]. Double inhibition experiments [20] in the presence of the two inhibitors (MgADP plus adenosine) were also carried out. Thiol reactivity studies. Kd values of ligand binding to PGK was determined on the basis of the protective effects of ligands against modification of the fast-reacting thiol groups. The rate of thiol modification by Nbs2 was followed at 412 nm (e412 = 14,150 M1 cm1) and analysed as described by Kova´ri et al. [8]. DSC experiments. The measurements were carried out in a MicroCal VP-DSC-type microcalorimeter (MicroCal Inc.) with a cell volume of 0.51 mL, at a constant scan rate of 60 °C/h. Tm values were determined in the presence of various concentrations of either D- or L-ADP. The data were analysed using MicroCal Origin 5.0. The maximum effects of ligands on the Tm values were determined by extrapolating the observed changes to infinite nucleotide concentrations using Eq. (2): T measured ¼ T min þ
ðT max T min Þ ½N K þ ½N
ð2Þ
where Tmin is measured in the absence of the nucleotide ligand, N, and Tmax is the extrapolated value at infinite [N]. SAXS measurements and data processing. Synchrotron radiation Xray scattering data were collected on the X33 beam line at the Hamburg EMBL Outstation [21] as described in Varga et al. [22]. Solution of hPGK (5–15 mg/mL, i.e. about 0.1–0.3 mM in the storage buffer), its binary complexes with 1,3-bPG (0.9 mM), D- or L-MgADP (2.2 mM), and the ternary complexes with 3-PG (5.7 mM) plus MgADP (2.2 mM) as well as with 1,3-bPG (0.9 mM) plus MgADP (2.2 mM for both enantiomers) were measured using a MAR345 Image Plate at a sample˚ , covering the detector distance of 2.4 m and wavelength of 1.5 A ˚ 1 (s = 4p sin (h)/k where momentum transfer range 0.012 < s < 0.45 A 2h is the scattering angle). The data were processed by PRIMUS [23] and the scattering patterns from the atomic models of the X-ray coordinates were computed using CRYSOL [24].
Results and discussion Characterisation of L-ADP as substrate or inhibitor: steady state kinetics
ð1Þ
ATP formation from 1,3-bPG and D- or L-MgADP PGK catalyses the reversible phospho-transfer between 1,3-bisphosphoglycerate (1,3-bPG) and MgADP: 1,3bPG + MgADP M 3-PG + MgATP. Usually the reaction is studied in the backward direction of glycolysis due to the sensitivity of 1,3-bPG towards hydrolysis. Here we followed the forward reaction using 1,3-bPG produced directly in the assay mixture. Fig. 1A shows the saturation curves with D- or L-MgADP. Thus, L-MgADP is almost as a good substrate for hPGK as the natural D-MgADP. The kcat and Km values with L and D enantiomers are 685 and 1085 s1, and 0.27 and 0.12 mM, respectively. The Km value for D-MgADP agrees well with that obtained earlier using the HK-G6PDH coupled assay [11].
where KI is the inhibition constant for Pi. KI value was obtained by a method involving HK and G6PDH [11] and was estimated to be 17.7 mM. Under the conditions used, GAP and NAD did not inhibit the PGK. The inhibitory properties of D- and L-MgADP were tested using 3-PG and MgATP as substrates. The reaction is again coupled to GAPDH reaction by reducing the product 1,3-bPG in the presence of NADH. The
D- and L-MgADP as inhibitors with MgATP and 3-PG as substrates In the PGK-catalysed reaction starting from 3-PG and MgATP, the product D-MgADP was shown to be a competitive inhibitor with respect to MgATP [25]. Fig. 1B compares the inhibitory effects of D- and
Km ¼
K app m KI ½Pi þ K I
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Fig. 1. Comparison of the kinetics of hPGK using L- and D-ADP either as substrate (A) or inhibitor (B–D). In (A) activities of 4 nM hPGK were recorded as a function of the L- (j) or D-ADP (d) concentration, in the presence of 15 mM MgCl2, 1 mM GAP, 0.5 mM NAD, 50 mM inorganic phosphate and the appropriate excess of GAPDH. The curves were fitted by the Michaelis–Menten equation. In (B–D) the PGK activity was monitored using MgATP and 3PG as substrates. The experiments were carried out with 8.8 nM (B) or 26.6 nM (C,D) hPGK in the presence of 10 mM (B) or 0.5 mM (C,D) 3-PG. All mixtures contained 0.5 mM ATP and 12.5 mM MgCl2. Activities were determined as a function of the L- (j, h) or D-MgADP (d, s) concentration. In (C,D) the inhibition was measured both in the absence (j, d) and in the presence of 20 mM adenosine (h, s) and plotted according to the model of Yonetani and Theorell [20].
L-MgADP
on this reaction. The Ki values for D- and L-MgADP are 35 and 63 lM, respectively. Thus, the relative values of the Ki resemble those of the Km values and similarly indicate a slight weakening of the interactions of L-MgADP with hPGK compared to those of D-MgADP. Double inhibition experiments To explore whether both D- and L-MgADP compete for the same adenosine-binding pocket that is located in the Cdomain of PGK, double inhibition experiments, similar to those published earlier with D-MgADP [9], were carried out in the presence of either D- or L-MgADP and adenosine. The Yonetani–Theorell plots [20] in Fig. 1C and D show parallel lines, indicating that the inhibitory effect of either D- or L-MgADP and adenosine are exclusive. Thus, it appears that D- and L-MgADP compete for the same adenosine site of PGK.
Effect of Mg2+ on the interactions of L- and D-ADP with hPGK: equilibrium studies For D-ADP binding the strengthening effect of Mg2+ is well documented [25,26]. Comparison of the binding constants of Mg2+-free- and MgADP showed an interesting difference between the two enantiomers, as revealed by thiol-reactivity studies (cf. below). The interaction of 2+ L-ADP with hPGK is much less strengthened by Mg (6-fold decrease in Kd, cf. Table 1) than that of D-ADP (at least 20-fold decrease in Kd [25]). The high-resolution crystal structure provides an explanation for the strengthening effect of Mg2+: the metal ion interacts with both the a- and b-phosphates of ADP as well as with the conserved Asp 374 located in helix 13 between the two domains [5]. Thus, the above differences may indicate different types of interactions of D- and L-ADP with hPGK through Mg2+.
A. Varga et al. / Biochemical and Biophysical Research Communications 366 (2008) 994–1000 Table 1 Dissociation constants (Kd) for the D- and L-enantiomers of ADP with or without Mg2+ Enantiomeric form
Ligand
Kd (lM)
D
ADP MgADP
590 ± 200 24 ± 2
L
ADP MgADP
410 ± 130 71 ± 18
The Kd values were determined using the method thiol reactivity, described in Materials and methods.
Effect of D- and L-ADP on the PGK conformation: biophysical studies Protective effect of D- and L-ADP against modification of the reactive thiols of PGK In a previous study, we showed that the reactivity of the two thiol groups located in helix 13 are sensitive to the presence of the substrates of PGK, in particular MgADP. Furthermore, MgADP has a much larger protective effect against thiol modification than Mg2+-free ADP [9], in accordance with the crystallographic data, showing ligation of the phosphates of ADP through Mg2+ to Asp 374 in helix 13 [5]. This interaction with MgADP makes helix 13 highly ordered as reflected by the low crystallographic average B-factor values for the main chain atoms of helix 13 [5,8]. Since ordering of helix 13 results in the shielding of the two reactive thiols and reduces their reactivity [8], testing of thiol reactivity is a sensitive tool for detection of substrate-caused local conformational changes. Here, the effects of L-ADP and L-MgADP were tested in the same way. The rate constants of thiol modification in the absence and presence of saturating amounts of D- or L-MgADP were found to be 1800 ± 22 and 30 ± 10 M1 s1, respectively (data not illustrated). This suggests that the binding modes of the phosphates of the MgADP enantiomers are approximately the same relative to the position of helix 13 and that both ligands cause similar local conformational changes around the reactive thiols. From dependences of the rate constants of thiol modification on the concentration of the nucleotides, their Kd values were also obtained and are given in Table 1. From the values we conclude that in spite of their similar effects on the conformation of helix 13, the interaction with Asp 374 (through Mg2+) is possibly weaker in case of L-MgADP. Protective effect of the nucleotides against unfolding Previous DSC studies showed that the substrates protect PGK against heat denaturation [9,27]. Here again, MgADP exerted much larger protection than Mg-free ADP (Fig. 2). From the experiments at different concentrations of L-MgADP, we could extrapolate to Tm at infinite concentration of the nucleotide. The extrapolated Tm value (59.5 ± 0.1 °C) is very similar to that obtained with D-MgADP. Thus, in spite of its mirror image conforma-
997
tion, by this method L-MgADP affects the overall conformation of PGK similarly to D-MgADP. Substrate-induced domain closure as detected by SAXS PGK is a monomer protein that contains two globular domains. The N-terminal domain binds 1,3-bPG or 3-PG and the C-terminal one contains the nucleotide-binding site. In the apoenzyme, the substrate binding sites are too far apart to allow phospho-transfer but upon the binding of both substrates an extensive ‘‘hinge binding motion’’ occurs that leads to the approximation of the sites [6,7,22]. In the latter work we have demonstrated that the scattering curves of substrate-free hPGK and its various binary and ternary enzyme-substrate complexes in solution correlate well with the theoretical curves calculated from the respective crystal structures, supporting the occurrence of domain closure in the ternary complexes [22]. Thus, the presence of both substrates is required for PGK domain closure. To determine whether or not L-MgADP induces domain closure, we carried out SAXS measurements on the binary EÆL-MgADP and the ternary EÆL-MgADPÆ1,3bPG and EÆL-MgADPÆ3-PG complexes. Thus, in addition to the latter non-functional ternary complex (investigated previously with D-MgADP) here we also studied the functioning L-MgADPÆ1,3-bPG.1 Control measurements were also carried out on the substrate-free PGK and on its binary complex with 1,3-bPG. The importance of the latter SAXS measurements is underlined by the fact that no crystal structure of PGKÆ1,3-bPG is available. The scattering curves obtained are shown in Fig. 3. The deviation of the experimental curves (dots with error bars) from the theoretical ones (continuous lines calculated from various open and closed crystallographic models) is expressed numerically by the discrepancy (v2) values (Table 2). The data show that the substrate-free enzyme exhibits a completely open structure. The binary complexes with LMgADP and D-MgADP gave the best agreement with the open crystal structure of the binary complex with MgATP [9]. The binary complex with 1,3-bPG agrees better with the partially closed conformation observed in the crystal structure of the binary complex with 3-PG [4]. The scattering curves of both functioning and non-functioning ternary complexes including L-MgADP (Fig. 3A) correlate well with the ones computed from the closed crystal structures, Thermotoga maritima [6] and Trypanosoma brucei [7]
1
Although the chemical equilibrium of either D- or L-MgADP phosphorylation reaction favours the formation of MgATP and 3-PG, but due to the high enzyme concentration (0.1–0.3 mM) applied and the extremely strong binding of the small molar excess (0.9 mM) of 1,3-bPG, the value of Kint, i.e. the equilibrium between the bound substrates and products possibly greatly differs from the above equilibrium [28,29] and the amounts of bound L-MgADP and.1,3-bPG are not negligible during the experiment. At any case, the data obtained starting from the complex with L-MgADP and 1,3-bPG, are characteristic of the average of the functioning ternary complexes, containing bound L -nucleotides (L-MgADP.1,3-bPG and L-MgATP.3-PG).
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Fig. 2. Effect of L- and D-MgADP on the thermal stability of hPGK. DSC transition curves (A) were determined in the absence (black dashed line) or in the presence of D-ADP (grey dashed line), D-MgADP (grey solid line) or L-MgADP (black solid line). The melting temperatures were plotted against the L(h) or D-MgADP (s) concentration (B). The free Mg2+ concentration was 2 mM. The solid lines represent the best fits of the experimental data given by Eq. (2).
Fig. 3. Experimental and calculated SAXS scattering curves for PGK obtained with L-MgADP (A) and D-MgADP (B). The experiments were carried out in the absence of substrates (1), or in the presence of 2.2 mM ADP plus 12.5 mM MgCl2 (2), 0.9 mM 1,3-bPG (3), 2.2 mM ADP, 12.5 mM MgCl2 plus 0.9 mM 1,3-bPG (4) or 2.2 mM ADP, 12.5 mM MgCl2 plus 5.7 mM 3-PG (5).
PGKs. The equivalent non-functioning ternary complex with D-MgADP and 3-PG was investigated previously and was found to be between the crystal structures of the binary complex with 3-PG (v2 = 2.049) and of the much better closed Thermotoga maritima PGK ternary complex (v2 = 2.018)) [22]. This marginal difference is, however, at the accuracy limit of the data. The present data provided similar results, not only for the non-functioning . D-MgADP 3PG, but also for the functioning . D-MgADP 1,3-bPG ternary complex (Fig. 3B and Table
2). Thus, we can conclude that, as expected, the ternary complexes, containing D-MgADP, possess largely closed conformations. Taken together, the unnatural L-MgADP in its functional ternary complex induces domain closure of hPGK, in agreement with the present kinetic results. In summary, hPGK can accommodate the mirror image L-enantiomer of MgADP into its nucleotide-binding site and can phosphorylate it, almost as effectively as the natural D-enantiomer. The fine structural details of the interactions of the two substrates (e.g. the interaction with Mg2+)
A. Varga et al. / Biochemical and Biophysical Research Communications 366 (2008) 994–1000
999
Table 2 Comparison of SAXS experimental parameters with those of the crystallographic models SAXS experiments
0
Discrepancy v2 between the scattering from crystallographic models and experimental dataa
Investigated hPGK complexes
) Rg (experimental) (A
Open crystal structures
GNOM method
Guinier method
Pig PGK (no ligand)
No ligand
23.9 ± 0.5
24.2 ± 0.1
1.91
3.72
2.43
2.07
6.02
6.64
Binary 1,3-bPG L-MgADP D-MgADP
23.7 ± 0.6 23.9 ± 0.6 23.7 ± 0.7
24.0 ± 0.2 24.1 ± 0.2 23.8 ± 0.2
1.71 2.38 1.50
2.59 2.80 1.51
2.00 1.70 1.48
1.58 1.93 1.62
3.32 3.02 1.91
3.64 3.71 2.23
22.7 ± 0.5 22.5 ± 0.5 23.6 ± 0.6 23.8 ± 0.5
22.9 ± 0.2 22.7 ± 0.2 23.9 ± 0.2 24.1 ± 0.2
1.58 1.91 1.76 1.43
1.70 1.88 1.83 1.61
1.58 1.82 1.54 1.38
1.55 1.81 1.52 1.35
1.59 1.80 1.58 1.39
1.42 1.73 1.69 1.79
24.25 43.7
24.34 43.2
24.02 43.6
23.97 43.8
23.26 43.7
22.64 45.3
Bs PGK MgADP binary
Pig PGK MgATP binary
Partially closed
Closed crystal structures
Pig PGK 3-PG binary
Tm PGK ternary1c
Tb PGK ternary2c
Ternary L-MgADPÆ1,3-bPG L-MgADPÆ3-PG D-MgADPÆ1,3-bPG D-MgADPÆ3-PG 0
) Rg (theoretical)b (A Molecular massb (kDa) a b c
The minimum values of discrepancy (in bold) indicate the best correlation between SAXS data and crystallographic model. Radius of gyration and molecular mass of the high-resolution models as retrieved from the PDB. Ternary1 and ternary2 denote MgAMP-PNPÆ3-PG and MgADPÆ3-PG ternary complexes, respectively.
in the enzyme active site are different for the D- and L-enantiomers, but these differences do not prevent the occurrence of the catalytic cycle. Based on our results, hPGK can be considered as a promising candidate for assisting in vivo phosphorylation of the pharmacologically potent L-nucleoside analogues with antiviral effects. Acknowledgments The paper was prepared within the framework of the Hungarian-French Intergovernmental Scientific and Technological Cupertino Program of the Hungarian Foundation of Research and Innovative Technology (OMFB00493/2007) as well as by Egide (Balaton program No. 14100ZF). The financial support by OTKA NI 61915 from the Hungarian National Research Foundation as well as the travelling grants for B.F., A.V. and Sz. J. by European Community-Research Infrastructure Action under the FP6 ‘‘Structuring the European Research Area Programme’’ (No. RII3-CT-2004-506008) are also gratefully acknowledged. D.S. and P.K. acknowledge support from the EU Design Study ‘‘SAXIER’’, contract no. 011934. A.V. was supported by a short term FEBS fellowship. References [1] P. Krishnan, Q. Fu, W. Lam, J.Y. Liou, G. Dutschman, Y.C. Cheng, Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of L-nucleoside analog diphosphates by 3phosphoglycerate kinase, J. Biol. Chem. 277 (2002) 5453–5459. [2] S. Gallois-Montbrun, A. Faraj, E. Seclaman, J.P. Sommadossi, D. Deville-Bonne, M. Ve´ron, Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs, Biochem. Pharmacol. 68 (2004) 1749–1756. [3] C. Mathe´, G. Gosselin, L-nucleoside enantiomers as antivirals drugs: a mini-review, Antiviral Res. 71 (2006) 276–281.
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