Báo cáo khoa học: Enzymatic properties of the lactate dehydrogenase enzyme from Plasmodium falciparum pdf

Enzymatic properties of the lactate dehydrogenase enzyme from Plasmodium falciparum Deborah K Shoemark1, Matthew J Cliff2, Richard B Sessions1 and Anthony R Clarke1 Department of Biochemistry, University of Bristol, UK Molecular Biology and Biotechnology Department, University of Sheffield, UK Keywords kinetic; lactate dehydrogenase; malaria; mechanism; Plasmodium falciparum Correspondence D K Shoemark, Department of Biochemistry, School of Medical Sciences, University Walk, Clifton, Bristol BS8 1TD, UK Fax: +44 117 9288274 Tel: +44 117 9288595 E-mail: deb.shoemark@bris.ac.uk (Received 24 January 2007, revised 13 March 2007, accepted 23 March 2007) The lactate dehydrogenase enzyme from Plasmodium falciparum (PfLDH) is a target for antimalarial compounds owing to structural and functional differences from the human isozymes The plasmodial enzyme possesses a five-residue insertion in the substrate-specificity loop and exhibits less marked substrate inhibition than its mammalian counterparts Here we provide a comprehensive kinetic analysis of the enzyme by steady-state and transient kinetic methods The mechanism deduced by product inhibition studies proves that PfLDH shares a common mechanism with the human LDHs, that of an ordered sequential bireactant system with coenzyme binding first Transient kinetic analysis reveals that the major rate-limiting step is the closure of the substrate-specificity loop prior to hydride transfer, in line with other LDHs The five-residue insertion in this loop markedly increases substrate specificity compared with the human muscle and heart isoforms doi:10.1111/j.1742-4658.2007.05808.x The lactate dehydrogenase enzyme from the parasite causing cerebral malaria, Plasmodium falciparum, is currently the subject of efforts to find alternatives to established drug regimens which suffer increasingly from problems of resistance and side-effects [1] This enzyme (PfLDH) catalyses the final step in the glycolytic pathway upon which the parasite relies during its anaerobic erythrocytic stages of development within the human host The natural product gossypol, derived from the cotton seed plant, is a known inhibitor of dehydrogenases Inhibition of PfLDH by gossypol derivatives has proved parasiticidal in vitro [2] and the search for specific inhibitors is underway [1,3] The enzyme PfLDH differs from the human isozymes in several important structural and kinetic features, among which is the possession of a five-residue insertion in the substrate-specificity loop [4] The fact that the enzyme has active-site properties that differ substantially from those of human LDHs implies that it might be possible to design selective inhibitors that could preferentially target the parasitic enzyme However, it is a considerable advantage in the selective targeting of an enzyme to have a firm grounding in both the structural and functional characteristics, the latter being useful in providing a basis for quantifying the effects of inhibitors on the enzyme To elucidate its functional characteristics, we have performed a mechanistic analysis using steadystate kinetics, equilibrium binding measurements and transient kinetic techniques It has been hitherto assumed that PfLDH follows the same kinetic mechanism as other LDHs In these experiments, we define the steady-state kinetic mechanism and associated rate constants in the forward and reverse directions, the coenzyme binding affinities and the nature of the rate-limiting step In addition, the effect of the unusual loop structure on substrate specificity is examined Abbreviations BsLDH, LDH enzyme from Bacillus stearothermophilus; FRET, fluorescence resonant energy transfer; LDH, lactate dehydrogenase; PfLDH, LDH enzyme from Plasmodium falciparum; TgLDH, LDH enzyme from Toxoplasma gondii 2738 FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS D K Shoemark et al Kinetic characterization of Pf LDH Results Product inhibition and binding order in the enzyme mechanism: determining the overall steady-state mechanism Substrate inhibition Figure shows the kcat value taken from the initial velocity of the reaction as a function of pyruvate concentration and near-saturating levels of NADH (kcat is used as it is independent of enzyme concentration) The fact that there is a reduction in velocity at high concentrations of pyruvate shows that the enzyme, in common with most lactate and malate dehydrogenases, is prone to substrate inhibition, although the magnitude of the effect is small The data were fitted to Equation (Experimental procedures) and reveal an inhibition constant (Ki) of 140 ± 18 mm, an apparent KM for pyruvate of 69 ± lm and a catalytic rate constant of 96 s)1 This value of Ki is high compared with that for human muscle LDH (4 mm) and the human heart enzyme (0.8 mm) [5] 100 kcat (s_1) 80 60 40 20 0 0.4 0.8 20 pyruvate (mM) 30 40 Fig Secondary plot of steady-state reaction velocities plotted as a function of pyruvate concentration Initial velocities of the enzyme reaction were measured under steady-state conditions, in varied concentrations of NADH and different fixed concentrations of pyruvate In these experiments, each initial rate measurement was repeated five times and the values averaged These data were fitted to the standard Michaelis–Menten equation to give values of kcat at a series of fixed pyruvate concentrations The curve shows these values for kcat versus pyruvate concentration fitted to Eqn in Experimental procedures, yielding a KM for pyruvate 69 ± lM, Ki 140 ± 18 mM (kcat is used as it is independent of enzyme concentration) Each point on the graph corresponds to five repeated measurements of initial rates at five different NADH concentrations fitted to yield kcat values with the standard error shown As there are nine of these points, the data correspond to 225 rate measurements An extensive steady-state analysis of the PfLDH reaction was performed to determine the basic mechanism, the catalytic rate constants for the forward and reverse reactions and KM values for pyruvate ⁄ lactate and NADH ⁄ NAD+, respectively Initially, a series of diagnostic steady-state experiments were designed to assign the general kinetic mechanism In these enzyme assays, NADH and pyruvate were used as the substrates and NAD+ or lactate as product inhibitors These studies were performed to test whether PfLDH has the characteristic mechanism for this class of dehydrogenases, i.e an ordered sequential binding system with NADH binding before pyruvate The manner in which products cause inhibition, i.e competitive, mixed or uncompetitive under certain experimental conditions are diagnostic of both the binding order and the extent to which the system exhibits rapid-equilibrium characteristics, i.e whether off-rates are much faster than turnover An initial set of four experiments used fixed, subsaturating concentrations of either substrate or cofactor with varied concentrations of the other The experiments were performed at different, fixed concentrations of either lactate or NAD+ A subsequent set of experiments was performed to see if saturating conditions could alleviate the effects on the apparent KM or kcat values An example of data from a steady-state product inhibition matrix is shown in Fig The inhibition patterns found in these experiments are summarized in Table They show that an ordered sequential bi-bi system in which NADH binds first is the appropriate mechanism for the enzyme The other six possible mechanisms are ruled out by the data in Table [6] Elucidation of steady-state kinetic constants The true KM value for pyruvate was determined using the secondary plot shown in Fig Here the apparent KM for pyruvate is plotted as a function of the concentration of NADH and fitted to Equation (see Experimental procedures) The plot shows a plateau at a value of about 55 ± lm, the true KM for the substrate Fig shows a secondary plot in which kcat values for these data sets were plotted as a function of the NADH concentration and fitted to the Michaelis– Menten equation The plot yields a value for the maximal catalytic rate constant of the reaction of 96 s)1 and a value for the KM for NADH of 11 ± lm FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS 2739 Kinetic characterization of Pf LDH D K Shoemark et al 10 KM apparent pyruvate (mM) 0.12 -0.02 0.08 0.06 0.04 0.02 -0.01 0.01 1/pyruvate (μM-1) 0.02 0.05 0.03 Fig Example plot of data from the steady-state product inhibition matrix, generating one piece of the information in Table This example shows a Lineweaver–Burk plot of rates under conditions of subsaturating NADH and varied pyruvate in the presence of different fixed lactate concentrations s, zero lactate; j, 50 mM lactate; n, 75 mM lactate; *, 100 mM lactate Each point on the graph represents an average of five measurements The kinetic constants describing the reaction in the other direction, with NAD+ and lactate as substrates, were determined at a physiologically relevant pH (pH 7.5) and all steady-state constants are given in Table In this study, for ease of purification and stability, a histidine-tagged version of PfLDH was used (see Experimental procedures) [7] To assess any effect of this tag on the catalytic function of the enzyme, equivalent experiments to those described above were performed with the wild-type enzyme The KM for NADH, the KM and Ki for pyruvate and kcat for both wild-type and His-tagged enzymes were measured These yielded the same constants, within error, as those Table Pattern of product inhibition in the steady state In order to elucidate the basic kinetic mechanism for PfLDH, the pattern of product inhibition was determined using NADH and pyruvate as substrates and either NAD+ or lactate as inhibitors For these diagnostic purposes, the reactions were carried out at two set concentrations of NADH, subsaturating (i.e KM · ¼ 10 lM) and saturating (i.e KM · 20 ¼ 200 lM) The pattern of inhibitory behaviour shown in the table is exactly that expected for an ordered bi bi kinetic mechanism with the coenzyme binding first [6] Product Subsaturating substrate Saturating substrate Substrate varied Lactate NAD+ Lactate NAD+ Mixed Mixed Mixed Competitive Mixed Not inhibited Uncompetitive Competitive Pyruvate Pyruvate NADH NADH 2740 0.1 0.1 NADH (mM) 0.15 0.2 Fig The secondary plot of apparent KM for pyruvate plotted against NADH concentration showing the true KM for pyruvate Initial velocities were measured in five different NADH concentrations and varied pyruvate Each measurement was repeated five times and averaged; this graph represents 125 measurements From a standard Michaelis–Menten fit, the data revealed apparent KM values for pyruvate for each NADH concentration (the error bars on the graph pertain to these fits) These apparent KM values for pyruvate were then plotted against the corresponding NADH concentration The data were fitted to Eqn in Experimental procedures and show the true KM for pyruvate; 55 ± lM is found at infinite NADH concentrations seen here as the plateau This behaviour also indicates that the system is an ordered sequential bireactant system with pyruvate binding subsequently to NADH [6] 100 80 60 kcat (s-1) 1/v (min/ΔA) 40 20 0 0.04 0.08 0.12 0.16 0.2 NADH (mM) Fig The secondary plot of steady-state reaction velocities plotted as a function of NADH concentration Data from the same set of experiments as described for Fig was used This time the secondary plot shows steady-state values for the fitted kcat measured with varied pyruvate at different fixed NADH concentrations The re-plotted data were fitted to the Michaelis–Menten equation to yield the KM for NADH as 11 ± lM and the kcat 96 s)1 FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS Kinetic characterization of Pf LDH Table Kinetic constants for the reduction of pyruvate and the oxidation of lactate at pH 7.5 Substrate ⁄ cofactor KM (lM) kcat (s)1) kcat ⁄ KM (s)1Ỉ M)1) NADH Pyruvate NAD+ Lactate 11 55 180 47 96 96 40 40 6.7 · 106 1.7 · 106 2.2 · 105 850 ± ± ± ± 24 · 103 described above, hence the tag did not influence the kinetic behaviour of the enzyme to any measurable extent c orre c te d FRET signa l (a rbitra ry units) D K Shoemark et al 4 Equilibrium binding affinity of NADH pH dependence of substrate binding A characteristic of this family of dehydrogenases is the pH sensitivity of the KM values for pyruvate and lactate [8] These parameters are controlled by the protonation state of the active-site histidine residue, which serves as a proton donor–acceptor in the redox reaction Pyruvate binds only when the histidine is in the protonated state and lactate only when it is unprotonated To investigate the pK of this residue, the KM for pyruvate was determined as a function of pH and the data are shown in Fig The data was fitted to Equation (see Experimental procedures) and shows that the KM is controlled by a single ionizing group with a pK of 7.95 ± 0.08, similar to other lactate dehydrogenases of this mechanistic family [9] Transient kinetic properties of the enzyme: the single-turnover reaction Single-turnover experiments were carried out to help elucidate the nature of the rate-limiting step In such 10 12 14 16 18 20 22 24 26 28 Pf LDH (μM) Fig The fluorescent resonance energy transfer (FRET) titration to establish the Kd for NADH One micromolar additions of PfLDH were made to a cuvette containing 10 lM NADH in a SPEX Fluoromax spectrophotometer The absorption wavelength for tryptophan at 285 nm was used as the excitation wavelength and the emission wavelength of 450 nm for NADH was monitored Control experiments were carried out to correct for the inner filter effect of adding protein as described The data were fitted to the tight binding equation (Eqn 5; see Experimental procedures) and the NADH concentration was allowed to float Results showed the Kd for NADH is ± 0.8 lM and the fitted NADH concentration was 10.4 ± 0.9 lM (10 lM in cuvette) apparent KM pyruvate (mM) The binding of NADH to the active site of dehydrogenases is usually accompanied by a significant alteration in its fluorescence properties resulting from either a protection from solvent, collision quenching and ⁄ or a change in polarity of the environment of the fluorophore In the case of PfLDH, the signal change on binding to the active site was too small to be used as a reliable reporter of the formation of the binary complex As a result, fluorescent resonance energy transfer (FRET) from the indole to the dihydro-nicotinamide groups was used to measure the affinity for NADH The FRET data were fitted to Equation (see Experimental procedures) and are shown in Fig 5, yielding a Kd of 4.0 ± 0.8 lm 0.1 0.0 0.0001 0.001 0.01 [H+] (μΜ) 0.1 Fig A pH titration under steady-state conditions was carried out to determine the pKa of the ionizable group at the active site The KM for pyruvate was determined from rates measured for 8–10 different pyruvate concentrations in 200 lM NADH in the pH range 6–10 (the enzyme was unstable below pH 5.5) Each rate was repeated three times and averaged Shown here is the log variation in apparent KM (mM) for pyruvate versus log [H+] NB In pH terms the x-axis reads left to right pH 10–6 A four buffer system was used to minimize variables other than pH (see Experimental procedures) The data has been fitted to the equation KMapp ¼ KM(1 + Kh ⁄ [H+]) The Kh from the graph was 11 ± nM and equates to a pKa of 7.95 ± 0.08 for the ionizable group FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS 2741 Kinetic characterization of Pf LDH D K Shoemark et al (postmix) pyruvate This result rules out the possibility that there is a rate-limiting, or partly rate-limiting, step that occurs after the binding of NADH and before the association of pyruvate, i.e a structural rearrangement of the E–NADH binary complex These transient kinetic results therefore demonstrate that the major rate-limiting step or steps occur after the binding of pyruvate Rapid kinetics of the multiple-turnover reaction The result of a multiple-turnover experiment in which 200 lm NADH was mixed with 35 lm enzyme at a pyruvate concentration of mm is shown in Fig The reaction trace (Fig 8A) shows curvature in the initial milliseconds of the experiment, followed by a steady-state rate of about 75 s)1 per active site as shown by the linear regression The first turnover was A 0.3 Absorbance an experiment, the enzyme is mixed with one equivalent of NADH to form a binary complex in one syringe of the stopped-flow apparatus This solution is then challenged with pyruvate and the first-order, onenzyme conversion of NADH to NAD+ is recorded by monitoring the loss of absorbance at 340 nm In this way, the reaction is simplified as it is only the hydride-transfer chemistry itself, or a preceding conformational rearrangement that can limit the recorded rate constant The single-turnover data are shown in Fig 7, where the observed rate constant is plotted against the varied concentration of pyruvate The data are fitted to the Michaelis–Menten equation giving a maximum rate constant 130 s)1 and an apparent KM of 240 lm The maximum rate constant is significantly higher than the catalytic rate constant measured in the steady state, suggesting that some other process is partially limiting the steady-state reaction rate The experiment at mm pyruvate was repeated, reversing the mixing order In this case, 75 lm enzyme was challenged with mm pyruvate and 75 lm NADH giving a single turnover rate for mm pyruvate postmix of 116 s)1 (data not shown) This is a similar rate to that seen in the previous experiment, with a preequilibrated binary complex challenged with mm 0.2 0.1 120 100 0 0.05 kobs(s-1) B 60 20 0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 0.04 0.02 pyruvate (mM) Fig The secondary plot of single turnover rates as a function of pyruvate concentration fitted to the Michaelis–Menten equation The KM value for pyruvate under transient conditions was 240 lM, five times weaker binding than in the steady state and the kcat was faster, 130 s)1 compared to 96 s)1 in the steady state Each single turnover rate was measured under transient kinetic conditions with equimolar enzyme and NADH in one syringe challenged with increasing concentrations of pyruvate in the other Each of the measurements was repeated 10 times, averaged and fitted to a single exponential giving the rate constant at each concentration of pyruvate 2742 0.15 0.06 Absorbance 40 0.1 Time (s) 80 0.02 0.04 Time (s) Fig Multiple turnovers measured in the stopped flow apparatus NADH (200 lM) was mixed with enzyme (35 lM) and mixed with pyruvate (1 mM) The change in absorbance at 340 nm was measured An average of five transients was used for the fitting (A) shows the averaged data from the experiment with a linear fit to the steady-state rate of 75 s)1 (B) shows the initial 0.04 s of the data after subtracting the steady-state rate These data were fitted to a single exponential to give an initial rate of 134 s)1 in the approach to the steady-state rate FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS D K Shoemark et al Kinetic characterization of Pf LDH fitted to a single exponential with slope for subsequent turnovers removed (Fig 8B) This gave a first-order rate constant of 134 s)1 This experiment shows that there must be a process following hydride transfer that partially limits the steady-state catalytic rate Primary deuterium isotope effect Figure shows a comparison of the single-turnover reaction carried out with NADH and with 4R-NADD The observed kinetic isotope effect, KIE(obs), was approximately 1.2 (given by the ratio of the first-order rate constants) Previous data for this class of dehydrogenase enzymes show that the intrinsic kinetic isotope effect [KIE(int)] should be close to 2.7 [10] for a reaction in which hydride transfer is completely ratelimiting This value was extrapolated from data on a series of LDH mutants [10] A plot of kcat versus the observed kinetic isotope effect showed that as kcat tended to zero the KIE tended to 2.7 The value of 2.7 was taken to represent the maximal KIE for an LDH limited by hydride transfer Here, the observed value of 1.2 indicates that while there is a small component from hydride transfer in the rate limiting process (the value is greater than 1), there must also be a major contribution from a conformational change Rate constants for hydride transfer (k3H) and conformational change (k3C) were calculated using Equations and change absorbance 340nm -0.01 (Experimental procedures) and found to be 2000 s)1 and 160 s)1, respectively A likely candidate for this conformational change is movement of the substrate-specificity loop, as observed in other lactate dehydrogenases [10] This will be considered in more detail, in the context of crystal structures, in the discussion Alternative substrates The fact that there is a unique five-residue insertion in the active-site loop of the PfLDH enzyme raises the possibility that substrate specificity is different from the LDHs thus far investigated in detail, both eukaryotic and prokaryotic To investigate this possibility, the activity of the enzyme was tested with alternative substrates for comparison with other well-characterized LDHs; Table shows a summary of the results There was an approximately 10-fold decrease in PfLDH efficiency between pyruvate and a-ketobutyrate The presence of the extra methylene group of a-ketobutyrate results in a 10-fold increase in KM However, the presence of two extra methylene groups, compared with pyruvate, in a-ketovalerate results in a catastrophic decrease in enzyme efficiency For this substrate the KM is increased 2000-fold and the kcat decreased 200-fold compared with a 130-fold increase in KM for a-ketovalerate in BsLDH, which had just a five-fold decrease in kcat [10] The ability of the enzyme to reduce phenylpyruvate was also assessed Surprisingly, and unlike the case of other LDH enzymes of this family, we could detect no catalytic activity at all with this substrate -0.02 Testing for malate dehydrogenase activity -0.03 -0.04 -0.05 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 Time (s) Fig The kinetic primary isotope effect measured in the stopped flow apparatus In this experiment 75 lM enzyme was challenged with 75 lM 4R-NADD (top trace) and 75 lM NADH (bottom trace) The ratio of rates of the two single turnover events is 1.2 The ratio expected (observed kinetic isotope effect) for this class of enzymes in a process that is wholly rate-limited by hydride transfer is 2.7 [10] The rate of conformational change was calculated as 160 s)1 and the rate of hydride transfer as 2000 s)1 using the equations described in the primary deuterium isotope effect section of Experimental procedures Each transient is an average of 10 One of the more striking sequence differences between PfLDH and other LDHs of the same fold is the presence of a lysine residue at position 102 The presence of a positive charge in this position in the sequence is a possible characteristic of an enzyme that has malate dehydrogenase activity [11] Indeed, apparent activity is seen under standard steady-state conditions when oxaloacetate is used as the substrate in place of pyruvate In neutral solutions, oxaloacetate Table Kinetic constants for pyruvate, a -ketobutyrate and a-ketovalerate (values in parentheses are taken from reference [16]) Substrate KM (mM) kcat (s)1) kcat ⁄ KM (s)1ỈM)1) Pyruvate a-ketobutyrate a-ketovalerate 0.055 0.6 (0.47) 116 96 80 (180) 0.64 1.7 · 106 1.3 · 105 (3.8 · 105) 5.5 FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS 2743 Kinetic characterization of Pf LDH D K Shoemark et al decarboxylates rapidly to pyruvate, even in the absence of an enzyme We used proton NMR to determine the actual substrate responsible for activity Over a period of hours, peaks for NADH and oxaloacetate were replaced by those corresponding to NAD+, lactate and pyruvate At no time were peaks corresponding to malate observed This indicates that oxaloacetate decarboxylates rapidly in the presence of PfLDH under these conditions and the observed activity at pH 7.2 is due to turnover of the resulting pyruvate Discussion The general reaction mechanism of PfLDH is, by and large, similar to those of other LDHs of the nicotinamide-dependent type The reaction follows an ordered bi-bi kinetic pattern [6] with coenzyme binding first (see Fig 10) In addition, the steady-state constants (see Table 2) are very similar to those measured for structurally related counterparts with KM values for NADH and pyruvate being typically in the 10)5 and 10)4 m ranges, respectively, and those for NAD+ and lactate being in the 10)4 and 10)2 m ranges Similarly the steady-state catalytic rate constants in each direction are in keeping with other LDHs With regard to the nature of the rate-determining steps, conformational rearrangement is the predominant kinetic barrier in the single-turnover reaction, i.e in a process that can only be limited by a rearrangement of the ternary collision complex or by the rate of hydride transfer, the latter must be the more rapid, as shown by the relatively small primary kinetic isotope effect The rate-limiting conformational rearrangement in other LDHs is identified as the closure of an active-site loop triggered by substrate binding The function of this change in structure is to remove solvent from the catalytic site and bring the positive charge of Arg-109 into proximity, so that the carbonyl group of pyruvate can be strongly polarized Additionally, loop-closure enhances substrate selectivity by engulfing the pyruvate within a catalytic vacuole to maximize contact between substrate and enzyme The steady-state catalytic rate constant is slightly slower than that recorded for the single-turnover reaction, showing that some process that follows hydride transfer partially limits the steady-state reaction cycle This process must be a product-release step, either a rate of dissociation of lactate or NAD+ or the rate of loop opening after the hydride transfer reaction A consequence of this partial rate-limiting process is that the apparent Michaelis constant for pyruvate in the single-turnover reaction is higher than that recorded in the steady-state This phenomenon is due to a relatively slow product off-rate in the system as described above To illustrate this, if the binding of pyruvate to the E–NADH complex is a rapid equilibrium process, then the measured KM (KM¢) in the singleturnover reaction is simply equal to the Kd for the formation of the encounter complex However, in the steady state all the partially rate-limiting steps come into play and the true KM is given by: KM ¼ KM =1 ỵ k3C =k3H ỵ k3C =k4 ị where k3c represents the rate of the conformational change, k3H the hydride transfer and k4 the rate of the product-off step Hence, in these circumstances, the steady-state KM is expected to be smaller than the apparent KM measured in the single turnovers Furthermore, it is interesting to note that the fact that all three of the above rate constants are partially rate-limiting shows that the enzyme obeys the ‘Knowlesian’ principle that biological catalysts should evolve to have no single, dominant energy barrier [12] Rather, there is an evolutionary advantage in equalizing the energies of intermediate and transition states in the on-enzyme reaction pathway A major aim of these experiments was to elucidate unusual features of the enzyme that might distinguish it from other LDHs, particularly those of human origin, having confirmed the lack of malate dehydrogenase activity The principal differences in the kinetic Fig 10 Simplified schematic of the mechanism of PfLDH where k3H and k3C represent the rate constants for hydride transfer and conformational change, respectively, as calculated from isotopic effects 2744 FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS D K Shoemark et al constants of PfLDH compared with human LDHs (with native substrate and cofactor) are twofold Firstly, substrate inhibition of PfLDH (140 mm; in the direction pyruvate to lactate) is much weaker than that shown by the human heart and muscle isoforms by around 175- and 35-fold, respectively [5] Second, the binding of NADH to PfLDH is some 10-fold weaker than that shown by the human isoforms, hence Kd for PfLDH is ± 0.8 lm compared with 0.5 and 0.6 lm for the human heart and muscle enzymes, respectively [5] Both of these differences (raised Kd and Ki) appear to be largely attributable to the presence of leucine at 163 in PfLDH, a residue which is serine in all known LDHs that not possess the extra five residues in the substrate-specificity loop Crystal structures of holo-LDHs with serine at position 163 show that the hydroxy group of the serine side-chain is hydrogen bonded to the nicotinamide amide group of NADH, often via a water molecule Site-directed mutagenesis has been used to make the S163L variants of both human heart and muscle LDH isoforms [5] In both cases, substrate inhibition was removed (Ki > 500 mm) and the Kd for NADH was raised about 10-fold compared with the wildtypes Structural studies of ternary complexes of plasmodial LDHs [13,14] all show a displacement in the position of the nicotinamide ring when compared with all other ternary LDH structures, which is consistent with the presence of leucine rather than serine at position 163 Whilst the human S163L mutants show rather similar kinetic and binding parameters for NADH, the KM for pyruvate is raised by 40- to 200-fold We may speculate that the five-residue insertion in the PfLDH substrate-specificity loop compensates for the deleterious effect on the pyruvate binding site due to the S163L change (since the reaction mechanism is ordered bi-bi, the pyruvate binding site is only fully formed after NADH binding) Some supporting evidence for this hypothesis comes from a kinetic study in which the substrate-specificity-loop sequences from the broad-specificity ketoacid reductase, l-hydroxyisocaproate dehydrogenase (l-hicDH), and PfLDH were engineered into Bacillus stearothermophilus LDH (BsLDH), replacing the wild-type loop [15] The BsLDH construct containing the l-hicDH loop (a four-residue insertion compared with typical LDHs, e.g those from human and bacillus) had a KM for pyruvate of 42 mm, raised some 670-fold over wild-type BsLDH By contrast, the BsLDH construct possessing the substrate-specificity loop from PfLDH had a KM for pyruvate raised only 13-fold to 0.8 mm, despite this corresponding to a five-residue loop insertion with respect to wild-type BsLDH Kinetic characterization of Pf LDH A simple method to explore the size of the substrate binding site in a functional enzyme is to measure its ability to turn over larger substrate-like molecules This is straightforward in the case of LDHs as many compounds R-CO.CO2H (R ¼ methyl in pyruvate) are readily available The data in Table clearly show that, in the case of PfLDH, extending R from ethyl (i.e a-ketobutyrate) to n-propyl (i.e a-ketovalerate) causes a catastrophic fall off in the catalytic efficiency (kcat ⁄ KM) of nearly six orders of magnitude In the case of wild-type BsLDH, this change causes a loss in catalytic efficiency closer to three orders of magnitude compared with pyruvate Even more striking are the relative activities of this pair of enzymes towards phenylpyruvate This bulky substrate is turned over by BsLDH with a reasonable catalytic efficiency of 1.8 · 104 m)1Ỉs)1 [15], whilst no activity at all was detected with PfLDH either in this study (data not shown) and elsewhere [16] This behaviour may be contrasted with that of two lactate dehydrogenases present in the parasite Toxoplasma gondii that turn over phenylpyruvate at a comparable rate to pyruvate Recent structural work [17] has shown that TgLDH1 has a very similar structure to PfLDH, including the long substrate-specificity loop Both TgLDH enzymes contain another loop insertion (of two residues) between helices a-G2 and a-G3 and other changes in residue types lining the active site, any or all of these factors may be responsible for the activity shown by TgLDHs towards phenylpyruvate Consequences for drug design The intolerance of PfLDH towards larger substrates limits the possibilities for inhibitor design based on substrate or product (i.e pyruvate or lactate) analogues This observation is borne out by the recent development of a series of azole-based lactate analogues which are strong inhibitors of the oxidized binary complex of PfLDH and NAD+ [3] Attempts to elaborate these compounds to improve binding and specificity were unsuccessful, presumably due to the precise conformational requirements of the closed substrate-specificity loop The bi-bi mechanism, demonstrated in this paper, requires binding of NADH before substrate As both the NADH and the ordered substrate-specificity loop comprise part of the substrate-binding site, substrate analogues are not expected to bind tightly to the apoenzyme However, an inhibitor that competes with endogenous NADH will firstly benefit from the 10-fold weaker affinity of NADH for PfLDH compared with the human LDH enzymes Additionally, the differences in residues FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS 2745 Kinetic characterization of Pf LDH D K Shoemark et al lining the NADH binding site such as the switch of Ser to Leu at position 163 should be exploitable in drug design Finally, with respect to improving affinity, compounds could be targeted to the apoenzyme [18] Binding compounds across the substrate and coenzyme sites could increase the scope for elaboration The large surface exposed when the substrate-specificity loop is disordered, as seen in the apoenzyme crystal structure, affords the opportunity to design inhibitors that are not restricted by the limited space available in the closed-loop conformation of the protein Experimental procedures To assess substrate inhibition, the data for experiments in which pyruvate was varied were fitted to the following equation: v ẳ Vmax :S=ẵS ỵ KM ỵ S2 =Ki ފ ð1Þ where v is the initial steady-state reaction velocity, S is substrate concentration, KM is the Michaelis constant for pyruvate and Ki is the substrate-inhibition constant At substrate concentrations well below Ki, this equation reduces to the standard Michaelis–Menten equation All experiments to elucidate the steady-state mechanism were performed at pyruvate concentrations at least 50-fold lower than Ki This equates to a reduction in rate of less than 2% hence the following rate equations not account for inhibition by substrate Expression and purification Steady-state rate equations Six histidines were added to the C-terminus of the PfLDH gene by PCR without linker or cleavage sites The modified gene was inserted into the pKK vector and cloned into JM105 strain of Escherichia coli [4] Cells were harvested from overnight culture in 2xYT (yeast tryptone media) without the need for isopropyl-b-d-thiogalactopyranoside induction [7] Following sonication, cell debris was separated by centrifugation at 5000 g for 30 The supernatant was then applied to a Nickel-NTA agarose column (Qiagen, Crawley, UK) The enzyme was eluted in 250 mm imidazole, concentrated against polyethylene glycol 20K and dialysed into phosphate buffered saline (NaCl ⁄ Pi), 10% glycerol, mm EDTA and 10 mm dithiothreitol This protocol yielded pure enzyme at an average of 80 mg cellsỈL)1 Aliquots (100 lL) of enzyme were snap frozen in liquid nitrogen and stored at )80 °C The activity of the enzyme stored under these conditions remained constant within the time-scale of the experiments The concentration of enzyme used was assessed by Bradford assay and by absorbance at 280 nm where mgỈmL)1 corresponds to an absorbance of 0.42 for a cm path length Enzyme purity was assessed as the only visible band by SDS ⁄ PAGE Where used for comparison with the his-tagged enzyme, wild-type PfLDH was expressed from a pKK vector in JM105 cells incubated overnight in 2xYT and purified on an oxamate affinity column and eluted with NADH Concentration, dialysis and storage methods were the same as for the his-tagged enzyme The steady-state rate equation for an ordered bi-bi reaction in the absence of reaction products is shown below Steady-state kinetics Enzyme assays were carried out at 25 °C using a Perkin Elmer spectrophotometer with a perfused cuvette block Grade I NADH and NAD+ were purchased from Boehringer Mannheim (Mannheim, Germany; now Roche) and the buffers and substrates from Sigma (Gillingham, UK) The data were analysed using grafit software 2746 v=E0 ¼ ðk1 Ák2 k3 ẵNẵPị=C0 ỵ ẵNẵPCNP ỵ ẵPCP ỵ ẵNCN ị 2ị where k1, k2 and k3 are the forward rate constants for the binding of NADH, the binding of pyruvate and the catalytic rate constant, respectively; the concentrations of NADH and pyruvate are [N] and [P], respectively; the coefficients CO, CNP, CP and CN represent the groups of rate constants that are dependent on the subscripted substrates, e.g Co are those that are independent of substrate and CNP those dependent on both coenzyme and substrate, etc The component rate constants are as follows: Co ẳ k-1ặk-3 + k-1ặk-2, CNP ẳ k1ặk2, CP ẳ k2ặk3, and CN ẳ k1ặk-2 + k1ặk3 To determine the Michaelis constants for pyruvate in the steady state, velocities were determined at a series of coenzyme and substrate concentrations and the apparent KM for pyruvate [KM.pyr.(app)] was determined as a function of NADH concentration ([N]) Using Eqn as the parent equation, the secondary data were then fitted to the following relationship: KM:pyr:appị ẳ CO =CNP ỵ ẵNCN =CNP ị=ẵN ỵ CN =CNP ị 3ị where the y-value at infinite [N] is CN ⁄ CNP, which translates to (k3 + k-2) ⁄ k2 and represents the true KM for pyruvate The apparent kcat of the system [kcat(app)] was determined using pyruvate as the varied reactant The value of kcat.pyr.(app) was then determined at a series of fixed NADH concentrations ([N]) Again using Eqn as the parent equation, the secondary data were fitted to the following derived relationship: kcat:pyr:appị ẳ k1 k2 k3 =CNP ịẵN=CP =CNP ỵ ẵNị 4ị where k1ặk2ặk3 CNP ẳ k3 and CP CNP ¼ k3 ⁄ k1 The former is the true kcat and the latter the true KM for NADH Steady-state reactions were carried out at 25 °C in 50 mm tris ⁄ 50 mm KCl buffer at pH 7.5 FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS D K Shoemark et al Kinetic characterization of Pf LDH Proton NMR analysis of reaction products pH dependence For the alternative substrates in addition to spectrophotometric assays, 1H NMR was used to assign the products formed in the presence of oxaloacetate and NADH As oxaloacetate undergoes decarboxylation to pyruvate, NMR was used to determine whether the activity seen was due to the turnover of oxaloacetate to malate or pyruvate to lactate Pyruvate formation, due to spontaneous oxaloacetate decarboxylation at low pH, was minimized by adding two molar equivalents of NaOH to ice-cold buffer prior to the addition of solid oxaloacetic acid In this manner, a preparative reaction was set up with PfLDH (3 lm), oxaloacetate (5 mm) and NADH (5 mm) in NaCl ⁄ Pi ⁄ D2O (pH 7.2) and H NMR was used to follow product formation The pH titration experiments at 25 °C were carried as for other steady-state assays A four buffer system was used comprising 20 mm each of potassium acetate, 2-(cyclohexylamino)-ethanesulfonic acid (CHES), 2-amino2-(hydroxymethyl)-1,3-propanediol (Tris), 2,2-bis(hydroxymethyl)-2,2¢,2¢-nitrilotriethanol (Bis Tris) The pH of the buffer was adjusted by the addition of either HCl or NaOH to produce a range of pH values from to 10 Measurements were made in saturating NADH and varied pyruvate to determine the KM for pyruvate at different pHs The pKa of the ionizable group was determined by fitting data to Eqn Transient kinetics Transient kinetic data were collected using an SX.18 mV apparatus supplied by Applied Photophysics For the stopped-flow reactions, buffers comprised 10% glycerol, 50 mm phosphate, 150 mm NaCl, with mm EDTA and 10 mm dithiothreitol at pH 7.5 Reactions were carried out at 25 °C Enzyme solutions were made up as stocks in this buffer and lost no activity during 48 h Glycerol is known to slow the rate of loop closure for other LDHs so the difference between steady-state rates in each of the buffers was assessed There was a 5–10% reduction in kcat in the presence of 10% glycerol so that, within error, direct comparisons could be made between stopped-flow data and the steady state All NADH solutions were diluted in buffer from freshly thawed 0.25 m stocks made up in water and stored at minus 80 °C NADH or 4R-NADD was added to enzyme immediately before rates were measured KMappị ẳ KM ỵ Kh =ẵHỵ ị The primary deuterium isotope effect was measured in the SX.18 mV to determine the difference in single turnover rates achieved in the presence of NADH or 4R-NADD Mono-deuterated cofactor was enzymically produced by formate dehydrogenase (from Candida methylica) in the presence of NAD+ and deuterated formic acid (kindly donated by C M Eszes, University of Bristol, UK) Formate dehydrogenase catalyses the addition of hydride (deuteride) to the A face of NAD+ giving 4R-NADD This is the same hydride (deuteride) that is transferred from the A face of the cofactor to pyruvate during reduction to lactate catalysed by LDH [19] PfLDH (75 lm) with 75 lm cofactor was mixed with mm pyruvate (premix concentrations) From these measurements the rates of conformational change and hydride transfer were calculated using the equations below kh ẳ ẵkobs;NADH kobs;NADD 1=R 1ị=kobs;NADD =R Kobs;NADH =Rị kc ẳ kobs;NADH kh ị=kh kobs;NADH ị Equilibrium uorescence FRET reactions were measured in a Spex Fluoromax spectrophotometer Over a 1500-s time-base, additions of lm enzyme were made to a solution of 10 lm NADH The excitation wavelength was 285 nm and emission monitored at 450 nm Adding protein to the cuvette causes an inner filter effect To compensate, an identical experiment was set up using N-acetyltryptophanamide in place of enzyme The PfLDH data were then divided by the resulting linear change in fluorescence for N-acetyltryptophanamide Data were fitted to a tight binding equation with floating [NADH]: Signal ẳ initial ỵ fE ỵ N0 ỵ Kd ị ẵE ỵ N0 ỵ Kd ị2 4N0 E0:5 g=2N0 ÞÞÁamp ð5Þ where ‘initial’ is the starting fluorescence, ‘amp’ the amplitude of change, E is the concentration of LDH added and N0 is total concentration of NADH in the titration (6) (7) 8ị where kobsNADH ẳ observed rate constant with NADH ẳ (kcặkh) (kc + kh); kobs,NADD ẳ observed rate constant with 4R-NADD ẳ [kcặ(kh R)] kc(kh ⁄ R); R ¼ 2.7 (the basis for R-value explained in results section for the primary deuterium isotope effect [7]); kh ¼ rate constant for hydride transfer; and kc ¼ rate constant for conformational change References Mehlin C (2005) Structure-based drug design for Plasmodium falciparum Comb Chem High T Scr 8, 5–14 Royer RE, Deck LM, Campos NM, Hunsaker LA & Vander Jagt DL (1986) Biologically active derivatives of gossypol: synthesis and antimalarial activities of periacylated gossylic nitriles J Med Chem 29, 1799–1801 Cameron A, Read JA, Tranter R, Winter VJ, Sessions RB, Brady RL, Vivas L, Easton A, Kendrick H, Croft FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS 2747 Kinetic characterization of Pf LDH 10 11 D K Shoemark et al SL et al (2004) Identification and activity of a series of azole-based compounds with lactate dehydrogenasedirected anti-malarial activity J Biol Chem 279, 31429–31439 Bzik DJ, Fox BA & Gonyer K (1993) Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli Mol Biochem Parasitol 59, 155–166 Hewitt CO, Eszes CM, Sessions RB, Moreton KM, Dafforn TR, Takei J, Dempsey CE, Clarke AR & Holbrook JJ (1999) A general method for relieving substrate inhibition in lactate dehydrogenases Protein Eng 12, 491–496 Segel IR (1993) Enzyme Kinetics – Behaviour and Analysis of Rapid Equilibrium and Steady State Enzyme Systems Wiley Classics Library edn John Wiley and Sons Inc., Chichester, UK Shoemark DK (2000) The kinetic characterization of lactate dehydrogenase enzyme from Plasmodium falciparum PhD Thesis University of Bristol, UK Fersht A (1998) Structure and Mechanism in Protein Science: a Guide to Enzyme Catalysis and Protein Folding, 4th edn W H Freeman, New York, NY Read JA, Winter VJ, Eszes CM, Sessions RB & Brady RL (2001) Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase Proteins Struct Func Genet 43, 175–185 Wilks HM, Halsall DJ, Atkinson T, Chia WN, Clarke AR & Holbrook JJ (1990) Designs for a broad specificity keto acid dehydrogenase Biochem 29, 8587– 8591 Brown MW, Yowell CA, Hoard A, Vander Jagt TA, Hunsaker LA, Deck LM, Royer RE, Piper RC, Dame JB, Makler MT et al (2004) Comparative structural analysis and kinetic properties of the lactate dehydrogenases from the four species of human malarial parasites Biochemistry 43, 6219–6229 2748 12 Albery WJ & Knowles JR (1976) Evolution of enzyme function and the development of catalytic efficiency Biochemistry 15, 5631–5640 13 Winter VJ, Cameron A, Tranter R, Sessions RB & Brady RL (2003) Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus Mol Biochem Parasitol 131, 1–10 14 Dunn CR, Banfield MJ, Barker JJ, Higham CW, Moreton KM, Turgut-Balik D, Brady RL & Holbrook JJ (1996) The structure of lactate dehydrogenase from Plasmodium falciparum reveals a new target for antimalarial design Nat Struct Biol 3, 912–915 15 Hewitt CO, Sessions RB, Dafforn TR & Holbrook JJ (1997) Protein engineering tests of a homology model of Plasmodium falciparum lactate dehydrogenase Protein Eng 10, 39–44 16 Gomez MS, Piper RC, Hunsaker LA, Royer RE, Deck LM, Makler MT & Vander Jagt DL (1997) Substrate and cofactor specificity and selective inhibition of lactate dehydrogenase from the malarial parasite P falciparum Mol Biochem Parasitol 90, 235–224 17 Kavanagh KL, Elling RA & Wilson DK (2004) Structure of Toxoplasma gondii LDH1: active-site differences from human lactate dehydrogenases and the structural basis for efficient APAD+ use Biochemistry 43, 879–889 18 Conners R, Schambach F, Read JA, Cameron A, Sessions RB, Vivas L, Easton A, Croft SL & Brady RL (2005) Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase Mol Biochem Parasitol 142, 137–134 19 Dolphin D, Poulson R & Avramovic O (1987) Pyridine Nucleotide Coenzymes Vol II Part A, pp 258–260 Wiley-Interscience, Chichester, UK FEBS Journal 274 (2007) 2738–2748 ª 2007 The Authors Journal compilation ª 2007 FEBS ... polarity of the environment of the fluorophore In the case of PfLDH, the signal change on binding to the active site was too small to be used as a reliable reporter of the formation of the binary... that the KM is controlled by a single ionizing group with a pK of 7.95 ± 0.08, similar to other lactate dehydrogenases of this mechanistic family [9] Transient kinetic properties of the enzyme: the. .. concentrations of either substrate or cofactor with varied concentrations of the other The experiments were performed at different, fixed concentrations of either lactate or NAD+ A subsequent set of experiments