Báo cáo khoa học: Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation pot

Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation Marta Sousa Silva 1 , Anto ´ nio E. N. Ferreira 1 , Ana Maria Toma ´ s 2,3 , Carlos Cordeiro 1 and Ana Ponces Freire 1 1 Centro de Quı ´ mica e Bioquı ´ mica, Departmento de Quı ´ mica e Bioquı ´ mica, Faculdade de Cie ˆ ncias da Universidade de Lisboa, Portugal 2 ICBAS – Instituto de Cie ˆ ncias Biome ´ dicas Abel Salazar, Universidade do Porto, Portugal 3 Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal All trypanosomatids share two characteristics that set them apart from other eukaryotic cells. The first is the functional replacement of glutathione by N 1 ,N 8 - bis(glutathionyl)-spermidine (trypanothione) whereby most glutathione-dependent enzymes are replaced by trypanothione-dependent ones [1]. The second is the compartimentation of glycolysis, which occurs in a specific organelle, the glycosome [2]. These differences may be exploited in the development of novel thera- peutic strategies based on the disruption of trypano- thione-dependent biochemical processes and glycolysis inhibition, both essential for the survival of these intra- cellular parasites. An often overlooked aspect of glycolysis arises from the chemical instability of dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate [3]. In physiologic Keywords Leishmania; trypanothione; methylglyoxal; glyoxalase; modelling Correspondence C. Cordeiro, Centro de Quı ´ mica e Bioquı ´ mica, Departmento de Quı ´ mica e Bioquı ´ mica, Faculdade de Cie ˆ ncias da Universidade de Lisboa, Edifı ´ cio C8, Lisboa, Portugal Fax: +351 217500088 Tel: +351 217500929 E-mail: caac@fc.ul.pt http://cqb.fc.ul.pt ⁄ enzimol Note The mathematical model described here has been submitted to the Online Cellular Sys- tems Modelling Database and can be accessed free of charge at http://jjj.biochem. sun.ac.za/database/silva/index.html (Received 12 November 2004, revised 21 January 2005, accepted 28 February 2005) doi:10.1111/j.1742-4658.2005.04632.x The glyoxalase pathway of Leishmania infantum was kinetically character- ized as a trypanothione-dependent system. Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates. A K m of 0.253 mm and a V of 0.21 lmolÆmin )1 Æmg )1 for glyoxalase I, and a K m of 0.098 mm and a V of 0.18 lmolÆmin )1 Æmg )1 for glyoxalase II, were obtained. Modelling and computer simulation were used for evaluating the relevance of the glyoxalase pathway as a potential therapeutic target by revealing the importance of critical parameters of this pathway in Leishma- nia infantum. A sensitivity analysis of the pathway was performed using experimentally validated kinetic models and experimentally determined metabolite concentrations and kinetic parameters. The measurement of metabolites in L. infantum involved the identification and quantification of methylglyoxal and intracellular thiols. Methylglyoxal formation in L. infan- tum is nonenzymatic. The sensitivity analysis revealed that the most critical parameters for controlling the intracellular concentration of methylglyoxal are its formation rate and the concentration of trypanothione. Glyoxalase I and II activities play only a minor role in maintaining a low intracellular methylglyoxal concentration. The importance of the glyoxalase pathway as a therapeutic target is very small, compared to the much greater effects caused by decreasing trypanothione concentration or increasing methyl- glyoxal concentration. Abbreviations DHAP, dihydroxyacetone phosphate; GAP, D-glyceraldehyde-3-phosphate; Glx I, glyoxalase I; Glx II, glyoxalase II; HTA, hemithioacetal; MG, methylglyoxal; TFA, trifluoroacetic acid; T(SH) 2 , N 1 ,N 8 -bis(glutathionyl)-spermidine; SDL-TSH, S-D-lactoyltrypanothione. 2388 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS conditions, these trioses readily undergo an irreversible b-elimination reaction of the phosphate group from their common 1,2-enediolate form, forming oxopro- panal (methylglyoxal) [4]. Methylglyoxal is also formed as a by-product of the triose phosphate isomerase cata- lysed reaction [5] and in bacteria may be enzymatically synthesized from dihydroxiacetone phosphate by meth- ylglyoxal synthase (EC 4.1.99.11), an enzyme not found in eukaryotic cells [6–8]. Once formed, methyl- glyoxal irreversibly modifies amino groups in lipids, nucleic acids and proteins, forming advanced glycation end products [9]. It is therefore toxic, mutagenic and an inhibitor of glycolytic enzymes [10]. The glutathi- one-dependent glyoxalase pathway is the main detoxifi- cation system for methylglyoxal [11]. It first reacts nonenzymatically with glutathione, forming a hemithio- acetal that is isomerized to the thiol ester S-d-lactoyl- glutathione by glyoxalase I (Glx I; lactoylglutathione lyase, EC 4.4.1.5). S-d-Lactoylglutathione is then hydrolysed to d-lactate and glutathione by glyoxalase II (Glx II; hydroxyacyl glutathione hydrolase, EC 3.1.2.6) as shown in Fig. 1. Enhancing methylglyoxal formation or inhibiting its main catabolic pathway may lead to an increase of methylglyoxal concentration with harmful effects on trypanosomatids that might be exploited for therapeu- tic purposes. Little is known regarding methylglyoxal metabolism in trypanosomatids and the first reference to the presence of the glyoxalase pathway in Leishmania braziliensis dates from 1988 [12]. Only 16 years later was glyoxalase II characterized in Trypanosoma brucei [13]. In this case, lactoyltrypanothione was found to be a better substrate for this enzyme than S-d-lactoylgluta- thione (SDL-TSH), the substrate for all glyoxalase II enzymes known so far. In this work we investigated the kinetics of the glyoxalase pathway enzymes in L. infantum by time course analysis based on modelling and parameter fit- ting with a genetic algorithm. The best-fit parameters were used to set up a mathematical model of the path- way in L. infantum. Computer simulation of the sys- tem’s behaviour resulting from excursions around a reference state were performed to reveal the most sen- sitive points of the glyoxalase pathway, towards pos- sible pharmacological opportunities. The mathematical model described here has been submitted to the Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun. ac.za/database/silva/index.html free of charge. Results and Discussion The potential of the glyoxalase system as a possible therapeutic target relies on its role as the main cata- bolic pathway for methylglyoxal in eukaryotic cells. To cause damage to Leishmania, or to any other trypano- somatid, conditions must be sought that lead to an increase of methylglyoxal concentration. A quantitative analysis of the most critical parameters of the pathway regarding this goal requires the knowledge of the intra- cellular concentrations of all metabolites involved and a kinetic model that accurately describes the glyoxalase system in Leishmania. Methylglyoxal was identified in Leishmania infantum by HPLC and appears to be the only 2-oxoaldehyde detected. This metabolite is present, in early stationary phase cells, at a concentration of 9.67 pmol per 10 8 promastigotes. This low methylglyoxal concentration suggests that its formation in L. infantum is nonenzy- matic as observed in other cells [14,15]. To confirm this hypothesis, methylglyoxal synthase activity was assayed by measuring methylglyoxal formation from dihydroxyacetone phosphate (DHAP). When compar- ing the rates of methylglyoxal formation in the pres- ence and in the absence of L. infantum extract, no significant differences were found. DHAP forms methylglyoxal at a rate of 0.17 lmÆmin )1 and with L. infantum extract the rate was 0.18 lmÆmin )1 . Data- base mining of the L. infantum genome did not reveal any possible sequences for a methylglyoxal synthase gene. The low intracellular methylglyoxal concentration Thiol esther CH 3 O O H CH 3 OH O S R H CH 3 OH O OH H RSH D-Lactate Glutathione or trypanothione -SH group Hemithioacetal Methylglyoxal Dihydroxyacetone phosphate 3-P-1,2-enediol D-glyceraldehyde -3-phosphate O 3 POCH 2 O H OH H H OH O 3 POCH 2 OH O 3 POCH 2 OH O H H (non-enzymatic) Glyoxalase I Glyoxalase II (non-enzymatic) 2-2- 2- CH 3 S R O OH H Fig. 1. Methylglyoxal metabolism. Methylglyoxal is formed from the glycolytic intermediates dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP), and is dismutated to D-lactate by the glyoxalase pathway. R-SH represents thiol group(s) of reduced glutathione (GSH) or reduced trypanothione [T(SH) 2 ]. M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2389 and the absence of methylglyoxal synthase activity sug- gest that this metabolite is most improbably originated from this enzyme’s activity. Therefore, in our model, we considered only the nonenzymatic formation of methylglyoxal from DHAP and d-glyceraldehyde- 3-phosphate (GAP) (Fig. 2) using the steady state concentrations of these trioses as previously reported [16]. Concerning the intracellular low molecular mass thiols of L. infantum, at early stationary phase of growth, HPLC analysis of monobromobimane deriva- tives revealed the presence of GSH and T(SH) 2 at retention times of 13.6 and 21.2 min, respectively (Fig. 3B). T(SH) 2 was present at a concentration of 3.04 nmol per 10 8 promastigotes, while GSH concen- tration was 0.50 nmol per 10 8 promastigotes, a much lower value. Unidentified thiols (U marked peaks) were also shown to be present in this parasite, at retention times of 14.5 and 23.3 min (Fig. 3B). GSH is present at a molar ratio of 1 : 6 relative to trypan- othione, making T(SH) 2 a good candidate for repla- cing GSH in the glyoxalase pathway in L. infantum, as occurs in other enzymatic systems in trypanosom- atids. Substrate dependence of the glyoxalase enzymes was then evaluated in this parasite by initial rate ana- lysis. Using the methylglyoxal glutathione hemithioacetal as substrate, the kinetic parameters for L. infantum glyoxalase I, were a K m of 1.85 ± 0.35 mm and a V of 0.19 ± 0.02 lmolÆmin )1 Æmg )1 (Table 1). The K m for Glx I, using this hemithioacetal, is about five times higher than that described for all known glyoxalase I enzymes with the methylglyoxal glutathione hemithio- acetal as substrate [11]. Additionally, Glx II activity could not be detected in L. infantum using S-d-lac- toylglutathione as substrate, either by following its hydrolysis at 240 nm or by monitoring GSH formation at 420 nm with 5,5¢-dithiobis(2-nitrobenzoic acid), a more sensitive assay [17]. Given these results and the much lower concentration of GSH compared to T(SH) 2 , it is likely that trypanothione hemithioacetal and lactoyltrypanothione might be the physiological substrates for glyoxalase I and glyoxalase II in L. infantum, respectively. Indeed, the kinetic parame- ters for Glx I were a K m of 0.24 ± 0.04 mm and a V of 0.19 ± 0.02 lmolÆmin )1 Æmg )1 using methyl- glyoxal trypanothione hemithioacetal (Table 1). For Fig. 2. The glyoxalase pathway in Leishmania infantum. Reactions 1 and 2 correspond to the nonenzymatic (n.e.) formation of MG from dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3- phosphate (GAP). Reactions 3 and 4 correspond to the reversible reaction between MG and reduced trypanothione [T(SH) 2 ]. Reac- tions 5 and 6 are catalysed by Glx I and Glx II, respectively. Num- bered reactions are described in Table 3. A B Fig. 3. HPLC analysis of the glyoxalase pathway metabolites in Leishmania infantum promastigotes. (A) Analysis of 2-oxoalde- hydes, showing the presence of MG as 2-methylquinoxaline and the internal standard (IS, 1 l M 2,3-dimethylquinoxaline). Other peaks are due to the reagent. (B) Thiol analysis, as monobromobi- mane derivatives. Glutathione (GSH) and trypanothione (T(SH) 2 ) were identified. Peaks marked R are due to the derivatizing reagent monobromobimane, while U marked peaks are unidentified thiols. The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al. 2390 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS Glx II, the activity could be measured and we obtained a K m of 0.073 ± 0.020 mm and a V of 0.22 ± 0.0005 lmolÆmin )1 Æmg )1 with bis(lactoyl)trypanothione (Table 2). The kinetic constants for both enzymes are similar to those found for glutathione or trypanothi- one-dependent glyoxalase I and II in other systems (Tables 1 and 2) [13,18–20]. The determination of detailed rate laws for enzyme systems is very difficult, unless a very large number of experiments is performed. This is seldom possible with trypanothione-dependent enzymes, given the scarcity of this thiol. Initial rate analysis is also limited to the study of isolated enzymes and does not provide a good approach to understanding the kinetics of a metabolic pathway. A better strategy is the use of time course analysis, which requires fitting of a set of parameters from a system of ordinary differential equations that describe a given kinetic model to a set of concentration time courses. So far, this analysis of the glyoxalase pathway has only been performed in yeast [20]. The glyoxalase pathway enzymes catalyse irreversible reactions and can be considered as single substrate Michaelian enzymes [11,20]. When fitting a single- enzyme model for glyoxalase I (single substrate irreversible Michaelis–Menten) to time courses for lactoyltrypanothione concentration, only a poor fit was possible (Fig. 4A,A¢). Other rate laws were investigated as possible alternatives and again no better fitting was achieved (data not shown). As we could detect the activity of both enzymes with trypanothione derived substrates we next fitted a two-enzyme kinetic model (single substrate irreversible Michaelis-Menten). In this case an excellent fit was achieved (Fig. 4B,B¢) and the kinetic parameters for both enzymes were determined (Tables 1 and 2). This fit was obtained using only two progress curves corresponding to 0.14 mm and 0.27 mm hemithioacetal. The analysis was also per- formed with more than two curves and identical results were obtained. For Glx I we determined an apparent K m of 0.253 mm and an apparent V of 0.21 lmolÆ min )1 Æmg )1 (Table 1) while for Glx II a K m of 0.098 mm and a V of 0.18 lmolÆmin )1 Æmg )1 were deter- mined (Table 2). Other models were tested, namely gly- oxalase II inhibition by methylglyoxal trypanothione hemithioacetal, but the fitting was not improved (data not shown). A possible effect of competitive product inhibition on glyoxalase I was also investigated, but a worse fitting was obtained (Fig. 4C,C¢). A K m of 0.801 mm and a V of 0.5 lmolÆmin )1 Æmg )1 were deter- mined, markedly different from the ones estimated from initial rate and time course analysis using the two-enzyme model. Moreover, the obtained K i of 0.02 mm would imply that the enzyme should have an abnormally high affinity for the product. With our experimental conditions, where native enzymes are present at their relative activities with Table 1. Glyoxalase I kinetic parameters in Leishmania infantum and other cells. Glx I Substrate Initial rate analysis Time course analysis K m (mM) V (lmolÆmin )1 Æmg )1 ) K m (mM) V (lmolÆmin )1 Æmg )1 ) Leishmania infantum GSH 1.85 ± 0.35 0.19 ± 0.02 – – T(SH) 2 0.24 ± 0.04 0.19 ± 0.02 0.253 0.21 Plasmodium falciparum [19] GSH 0.77 ± 0.15 NC a –– Leishmania major [18] T(SH) 2 0.32 ± 0.03 NC a –– Saccharomyces cerevisiae [20] GSH 0.51 ± 0.06 NC b 0.62 ± 0.18 NC b a NC, not comparable (data from recombinant enzyme). b NC, not comparable (data from permeabilized cells). Table 2. Glyoxalase II kinetic parameters in Leishmania infantum and other cells. ND, not detected. Glx II Substrate Initial rate analysis Time course analysis K m (mM) V (lmolÆmin )1 Æmg )1 ) K m (mM) V (lmolÆmin )1 Æmg )1 ) L. infantum SDL-GSH ND ND – – SDL-TSH 0.073 ± 0.020 0.22 ± 0.0005 0.098 0.18 T. brucei [13] SDL-TSH 0.086 ± 0.004 NC a –– S. cerevisiae [20] SDL-GSH 0.32 ± 0.13 NC b 0.09 ± 0.05 NC b a NC, not comparable (data from recombinant enzyme). b NC, not comparable (data from permeabilized cells). M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2391 possible post-translational modifications preserved, we achieved a characterization of the glyoxalase system sufficient to elaborate a minimal model of its global kinetic behaviour (Fig. 2). A reference steady state was defined by the experimentally determined enzyme activities using time course analysis and the measured intracellular trypanothione concentration. The rate of methylglyoxal formation was calculated using the pre- viously determined triose phosphate concentrations [16] and rate constants [21]. When simulating the effects of changing glyoxalase I or glyoxalase II activities on methylglyoxal steady- state concentration, surprising results were obtained (Fig. 5A,B). To increase methylglyoxal concentration by about 50%, glyoxalase I activity must be decreased to 10% of its reference value (Fig. 5A). Varying glyoxalase II activity causes no noticeable change on the concentration of methylglyoxal within the tested range of variation (Fig. 5B). By contrast, methylglyoxal input and trypanothione concentration show a linear and an inverse hyperbolic effect on the steady-state concentration of methylglyoxal, respect- ively (Fig. 5C,D). In search for synergistic effects, the dependence of methylglyoxal steady-state concentration on the joint variations of two parameters at a time was also simu- lated (Fig. 6). Focusing on the glyoxalase activities, trypanothione concentration, and methylglyoxal for- mation rate as model parameters, there are six possible two-parameter combinations to be considered. Among these, a significant increase in methylglyoxal is only achieved when trypanothione concentration is decreased (Fig. 6A,B). The greatest effect is observed for the simultaneous increase of methylglyoxal forma- tion rate and decrease of trypanothione concentration. In all other combinations there is only a slight effect on methylglyoxal concentration suggesting that a signi- ficant increase of this metabolite would only be AA' BB' CC' Fig. 4. Time course analysis of the glyox- alase pathway in Leishmania infantum. Two concentrations of methylglyoxal trypanothi- one hemitioacetal were studied (0.14 and 0.27 m M). Lactoyltrypanothione concentra- tion was monitored at 240 nm. Experimental data (black line, A,B,C), fitting a single- enzyme model (blue line, A), fitting a two- enzyme model (red line, B) and fitting a single-enzyme model with competitive prod- uct inhibition (yellow line, C). The best fit for each model was obtained by least squares minimization using two time courses and a genetic algorithm to search the parameter space. Numerical solvers of ODE initial value problems and the genetic algorithms were implemented in the software package AGEDO. For each model, plots of residuals are shown in A¢,B¢ and C¢. The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al. 2392 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS possible for extreme modulations of enzyme activities. In particular, in the combinations involving the decrease of glyoxalase II activity the effect is equival- ent to the modulation of the other parameters alone, as shown in the combination involving glyoxalase I and glyoxalase II (Fig. 6C). The simulation results, based on experimentally determined parameters and a kinetic model of the AB DC Fig. 5. Sensitivity analysis of the glyoxalase pathway in Leishmania infantum. The effects of system parameters on the intracellular steady-state concentration of methylglyoxal were investigated by finite parameter chan- ges (between 0.05- and three-fold) around the reference steady state. All values are fold variations relative to the reference state (normalized values). System parameters were: glyoxalase I activity (A), glyoxalase II activity (B), methylglyoxal input (C), and initial trypanothione concentration (D). 0 20 40 60 80 1.0 1.5 2.0 2.5 3.0 0.2 0.4 0.6 0.8 1.0 MG MG input initial SH 0 20 40 60 80 0 20 40 60 80 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 MG GLX I initial SH 0 20 40 60 80 0 20 40 60 80 0.2 0.4 0.6 0.8 1.0 MG GLX I GLX II 0 20 40 60 80 0 20 40 60 80 0.2 0.4 0.6 0.8 1.0 1.0 1.5 2.0 2.5 3.0 MG GLX I MG input 0 20 40 60 80 AB C D 0.2 0.4 0.6 0.8 1.0 Fig. 6. Sensitivity analysis of the glyoxalase pathway in Leishmania infantum, studying the effects of two simultaneous system parameters on the intracellular steady-state concentration of methylglyoxal, by finite parameter changes (between 0.05- and onefold, except for MG input that was between one- and 3.5-fold) around the reference steady state. All values are fold variations relative to the reference state (normalized values). System parameters were: initial trypanothione concentration and methylglyoxal input (A), initial trypanothione concentration and glyoxalase I activity (B), glyoxalase II activity and glyoxalase I activity (C), methylglyoxal input and glyoxalase I activity (D). M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2393 pathway, clearly show that the glyoxalase enzymes are poor therapeutic targets. This view is supported by growth experiments with single gene deletion yeast mutants for glyoxalase I and II [21]. Both strains grow in d-glucose containing media in exactly the same way as the reference strain. Only when methylglyoxal is added to the growth medium at a concentration of 0.5 mm a slight reduction of growth rate is observed for the DGLO1 strain. Growth of the DGLO2 strain is not affected even in the presence of 1 mm of methyl- glyoxal. Moreover, glyoxalase II is absent in some mammals with no harmful consequences [22]. Methylglyoxal formation is nonenzymatic in eukary- otic cells and Leishmania is no exception. Its formation rate is dependent of triose phosphates concentrations and may be changed by controlling triose phosphate isomerase (TPI) activity, for a given glycolytic flux. In a case study of human TPI deficiency, increased con- centrations of DHAP and methylglyoxal were detected, related to mental illness [23]. Additionally, reduction of TPI activity in Trypanosoma brucei causes an inhibi- tion of growth, likely due to increased methylglyoxal formation [24]. A detailed kinetic and molecular char- acterization of L. infantum TPI may lead to the devel- opment of specific inhibitors granting a selective inhibitory effect that may prove to be useful against trypanosomatids. The intracellular concentration of trypanothione is another critical parameter that will lead to an increase of the steady-state concentration of methylglyoxal. Again, in the work with yeast referred to above, the most sensitive strain to methylglyoxal is the one lack- ing glutathione synthase I, DGSH1, with a lower intra- cellular GSH concentration [21]. In Trypanosoma brucei, trypanothione depletion results in growth arrest and increased sensitivity to oxidative stress [25]. Inhibi- tion of trypanothione biosynthesis most likely impairs several pathways vital to the survival of the parasite. Moreover, resistance to carbonylic stress caused by methylglyoxal will be compromised. From a practical point of view, trypanothione depletion might be achieved by inhibiting trypanothione synthetase the enzyme that in T. brucei, T. cruzi and L. major was shown to catalyse the formation of that thiol from spermidine and glutathione [26–28]. This enzyme, essential to T. brucei [29] and very likely to the other trypanosomatids, is considered one of the most prom- ising targets for chemotherapy. In summary, research efforts in search for more effective drugs against trypanosomatids have revealed important aspects of these parasites’ biochemistry. Effective therapies must rely on unique aspects such as glycolysis compartimentation and thiol metabolism. Trypanothione is essential for cell viability and plays a major role in the defence against oxidative stress caused by hydrogen peroxide and organic hydroper- oxides. It is also the physiological substrate of the gly- oxalase pathway, the main detoxification system for methylglyoxal and other 2-oxoaldehydes, arising from nonenzymatic reactions. As any prospects to fulfil this goal rely on increasing methylglyoxal concentration, our results clearly show that reduction of glyoxalase I or glyoxalase II activities will have only a slight to no effect, respectively, on steady-state concentration of methylglyoxal. On the contrary, focusing on increasing methylglyoxal forma- tion or reducing trypanothione concentration are more attractive approaches. In the case of trypanothione, a synergistic effect, whereby oxidative and carbonylic stresses are increased, may be achieved with lethal consequences to trypanosomatids. Experimental procedures Reagents and equipment S-d-Lactoylglutathione (SDL-GSH), yeast glyoxalase I (530–550 UÆmg )1 protein), bovine liver glyoxalase II (% 29 UÆmg )1 protein), N-ethylmaleimide, dithiothreitol, DHAP, methylglyoxal dimethylacetal, trifluoroacetic acid (TFA), monobromobimane, 1,2-diaminobenzene, 5,5¢- dithiobis(2-nitrobenzoic acid) and Coomassie Brilliant Blue G were purchased from Sigma Chemical Co (St Louis, MO, USA). 2,3-Dimethylquinoxaline was obtained from Aldrich. Reduced and oxidized glutathione (GSH and GSSG) were obtained from Boehringer Mannheim GmbH (Mannheim, Germany). Trypanothione disulfide (TS2) was purchased from Bachem. RPMI Medium was purchased from Gibco-BRL (Paisley, UK). Other reagents were of analytical grade and all solvents were of HPLC grade. A Beckman DUÒ (Fullerton, CA, USA) 7400 diode array spectrophotometer with a thermostated multicuvette holder, with stirring, was used for the determination of protein con- centration and to monitor enzyme activity. Centrifugations were performed in a refrigerated Eppendorf (Hamburg, Germany) 5804R centrifuge. Thiol determinations and methylglyoxal (MG) quantifications were performed in a Beckman Coulter HPLC coupled with a Jasco FP-2020 Plus (Tokyo, Japan) fluorescence detector. In these assays, a Merck LichroCART (Darmstadt, Germany) 250–4 (250 · 4 mm) column with stationary phase Merck LiChro- spher Ò (Darmstadt, Germany) 100 RP-18 (5 lm) was used. Preparation of metabolites High-purity MG was prepared by acid hydrolysis of meth- ylglyoxal dimethylacetal, in 10% (v ⁄ v) H 2 SO 4 , and purified The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al. 2394 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS by fractional distillation under reduced pressure in nitrogen atmosphere [30]. The solution obtained was calibrated with yeast Glx I and bovine liver Glx II. Oxidized glutathione (GSSG) and oxidized trypanothione (TS 2 ) were reduced with dithiothreitol, in the proportion of 1mm GSSG or TS 2 )3.2 mm dithiothreitol. The reaction was performed at 60 °C for 20 min, in a 1.5 mL reaction system, in 0.1 m potassium phosphate buffer, pH 6.8. SDL-TSH was prepared from reduced trypanothione and MG using yeast glyoxalase I. MG was added in excess (3.34 mm in a 2 mL reaction system), and the hemithio- acetal concentration was calculated using the value of 3.0 mm for the dissociation constant [31]. Glyoxalase I reaction was started by the addition of yeast Glx I. The formation of SDL-TSH was followed at 240 nm, and its concentration was calculated using a e 240 of 6.5 mm )1 Æcm )1 [13]. The enzyme was removed after completing the reaction using an Ultrafree-MC Filter 5KDa (Millipore, Billerica, MA, USA), and the recovered solution was used for the glyoxalase II activity assay. Leishmania infantum culture Promastigotes of Leishmania infantum clone MHOM ⁄ MA67ITMAP263 were grown in RPMI medium supple- mented with 10% fetal bovine serum, 2 mml-glutamine, 50 mm Hepes sodium salt (pH 7.4), 35 UÆmL )1 penicillin and 35 l g ÆL )1 streptomycin, at 25 °C [32]. Preparation of Leishmania infantum extracts Promastigotes of L. infantum at early stationary phase of growth (about 150 mL, containing approximately 10 9 cells) were washed twice in NaCl ⁄ P i , and suspended in 1 mL NaCl ⁄ P i . To prepare the protein extracts for enzyme assays, cells were submitted to eight freeze–thaw cycles (on ice and 50 °C) and the supernatant was recovered after centrifuga- tion at 10 500 g for 10 min. Protein concentration was quan- tified according to Bradford using BSA as the standard [33]. For thiol identification and MG quantification, cells were lysed and deproteinized with 0.5 m perchloric acid. The sus- pension was kept on ice for 10 min, vortexed for 2 min and centrifuged at 4 °C, 10 500 g, for 5 min. The recovered supernatant was immediately analysed or stored at )80 °C [14]. Thiol assay Intracellular thiols were derivatized with the fluorescent label monobromobimane and analysed by HPLC. The deri- vatization procedure was based on the methods described by Tang et al. [34] and by Ondarza et al. [35], with some modifications. A 100 lL aliquot of the L. infantum extract (containing 10 8 cells) was neutralized with KOH and centrifuged at room temperature for 3 min at 10 500 g. The reduction of oxidized thiols was performed with dithiothrei- tol at a final concentration of 0.4 mm in 0.5 m Tris ⁄ HCl pH 8.0, for 20 min at 60 °C. Monobromobimane (in aceto- nitrile) was added to a final concentration of 1 mm (200 lL reaction system) and the derivatization was carried out at 60 °C for 35 min, in the dark. Perchloric acid, at a final concentration of 0.5 m, was added to stop the reaction. Thiol standards GSH and T(SH) 2 were submitted to the same treatment. A 20 lL sample volume was injected. Elu- tion of bimane-derivatized compounds was monitored by fluorescence detection with excitation at 397 nm and emis- sion at 490 nm, using a binary gradient of acetonitrile with 0.08% (v ⁄ v) TFA (solvent A) and water with 0.08% (v ⁄ v) TFA (solvent B). The gradient program was: 0–5 min, 10% (v ⁄ v) solvent B isocratic; 5–35 min, 10–30% (v ⁄ v) solvent B; 35–40 min, 30–10% (v ⁄ v) solvent B. Separation was car- ried out at a flow rate of 1.0 mLÆmin )1 . GSH and T(SH) 2 were identified and quantitated by comparison with stand- ards. Thiol concentrations were calculated from calibration curves performed with known concentrations of monobro- mobimane-derived thiols. For control samples, thiols were blocked with 5 mm N-ethylmaleimide for 20 min at 60 °C before derivatization. Methylglyoxal assay Intracellular methylglyoxal was measured in L. infantum (100 lL extract) with a specific HPLC-based assay, by deri- vatization with 1,2-diaminobenzene and using 2,3-dimethyl- quinoxaline as internal standard [36]. Methylglyoxal synthase activity was assayed by measuring methylglyoxal formation from DHAP. The reaction occurred in 1 mL reaction volume, in 0.1 m potassium phos- phate buffer, pH 6.8, at 30 °C. DHAP was added to a 50- and a 100-lL aliquot of the L. infantum extract, to a final concentration of 1 mm. The reaction was stopped with the addition of perchloric acid to 0.5 m final concentration. Controls were performed without L. infantum extract and the rates of methylglyoxal formation compared. Methylgly- oxal was measured in all samples, at time zero and after 2.5 h of incubation, with the HPLC assay referred to above. Enzyme kinetic assays Enzyme activities were determined at 30 °C in a 2 mL reac- tion volume, in 0.1 m potassium phosphate buffer, pH 6.8. Magnetic stirring in the spectrophotometer cuvette was used to maintain isotropic conditions. The Glx I activity assay was based on the method des- cribed by Martins et al. [20] with some modifications. Glx I activity was assayed with GSH, with dithiothreitol reduced GSSG, and with reduced trypanothione [T(SH) 2 ], using MG in excess (3.34 mm). Initial concentrations of GSH and GSSG were calculated to give hemithioacetal concentra- tions from 0.16 to 3.8 mm. Initial concentrations of T(SH) 2 M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2395 were calculated to give substrate concentrations from 0.035 to 0.97 mm. Hemithioacetal concentration was calculated using the value of 3.0 mm for the dissociation constant [31], and its formation was followed for 20 min after the addi- tion of MG. Glyoxalase I reactions were started by the addition of the protein extract (15 lg of total parasite pro- tein) and the formation of SDL-GSH or SDL-TSH was fol- lowed at 240 nm. The concentration of these compounds was determined using a e 240 of 2.86 mm )1 Æcm )1 [37] and 6.5 mm )1 Æcm )1 [13] for the SDL-GSH and SDL-TSH, respectively. dithiothreitol does not interfere with Glx I assays. Glyoxalase II activity assay was performed using the commercially available SDL-GSH and SDL-TSH prepared from T(SH) 2 and MG using yeast glyoxalase I, as previ- ously described. Concentrations of SDL-GSH between 0.5 and 4 mm were used and SDL-TSH concentrations between 0.05 and 0.10 mm were prepared. The reactions occurred in the same conditions, and were started with the addition of protein extract (15 lg of total protein). The hydrolysis of both thiolesthers was followed at 240 nm. Glyoxalase II activity with SDL-GSH was also assayed by following GSH formation at 412 nm with 5,5¢-dithiobis(2-nitro- benzoic acid) [20]. Determination of kinetic parameters The kinetic parameters for glyoxalase I and II were deter- mined using two different approaches, initial rate analysis and time course analysis. Initial rate data were fitted to irreversible single substrate Michaelis–Menten models. Non-weighted hyperbolic regres- sion by the method of least squares was performed with the program HYPER (J. S. Easterby, University of Liverpool, UK; http://www.liv.ac.uk/$jse/software.html). In time course analysis the parameters were determined by minimization of the difference between experimental time course data and the corresponding values predicted by the solution of the differential equations derived from a mathematical model of the kinetic assay. In this analysis, different models were tested. In ‘single-enzyme model’, only the reaction of glyoxalase I with an irreversible Michaelis– Menten rate law was considered (Scheme 1). HTA SDL-TSH [] [] HTA HTA 1 1 1 + = m K V v In the ‘two-enzyme model’, the consecutive reactions of gly- oxalase I and glyoxalase II, both with irreversible Michaelis– Menten rate laws were considered (Scheme 2). [] [] HTA HTA 1 1 1 + = m K V v [] [] TSH-SDL TSH-SDL 2 2 2 + = m K V v HTA SDL-TSH In ‘single-enzyme model with product inhibition’, only the reaction of glyoxalase I was considered, with an irre- versible Michaelis–Menten rate law with competitive prod- uct inhibition (Scheme 3). HTA SDL-TSH [] [] [] HTA TSH-SDL 1 HTA 1 1 1 1 +         + = iP m K K V v The best fit for each model was obtained with the program AGEDO [38] using two time courses of SDL-TSH. Minimi- zation over the parameter space was performed using the genetic algorithm ‘differential evolution’ [39]. In each search, the best fit vector of kinetic parameters h was defined by the minimum of the objective function SS(h) given by Eqn (1): SS hðÞ¼ X p k¼1 X n k i¼1 X OBS k t i ðÞÀX SIM k t i hðÞ ÀÁ 2 Eqn ð1Þ In this equation, p is the number of time courses used in the analysis, n k is the number of points in time course k, X OBS k t i ðÞis the experimental value of the SDL-TSH for time Table 3. Rate equations and kinetic parameters of the glyoxalase pathway model. Rate equations are shown in Fig. 2. Kinetic models for the two enzymes were experimentally validated by time course analysis. Intracellular concentrations of methylglyoxal and trypano- thione were calculated using an estimate of the L. infantum cell volume of 75 lm 3 , based on cell measurement. Other constants and metabolite concentrations were from previously published works. Initial concentrations of MG, hemithioacetal and SDL-TSH were zero. Differential equations dMG ⁄ dt ¼ (v 1 + v 2 )–v 3 + v 4 dHTA ⁄ dt ¼ v 3 – v 4 – v 5 dSDLTSH ⁄ dt ¼ v 5 – v 6 dT(SH) 2 ⁄ dt ¼ – v 3 + v 4 + v 6 Rate equations v 1 ¼ k 1 GAP v 2 ¼ k 2 DHAP v 3 ¼ k 3 MG T(SH) 2 v 4 ¼ k 4 HTA v 5 ¼ V 5 HTA ⁄ (K m5 + HTA) v 6 ¼ V 6 SDLTSH ⁄ (K m6 + SDLTSH) Parameters k 1 ¼ 6.4 · 10 )3 min )1 k 2 ¼ 6.6 · 10 )4 min )1 k 3 ¼ 0.34 mM )1 Æmin )1 k 4 ¼ 1.01 min )1 V 5 ¼ 2 · 3.042 mMÆmin )1 V 6 ¼ 2 · 2.653 mMÆmin )1 K m5 ¼ 2 · 0.253 mM K m6 ¼ 2 · 0.0980 mM GAP ¼ 0.0072 mM DHAP ¼ 0.16 mM T(SH) 2 (at time zero) ¼ 2 x 0.45 mM The glyoxalase pathway in Leishmania infantum M. Sousa Silva et al. 2396 FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS course k at time t i , and X SIM k t i hðÞis the corresponding value predicted by the numerical solution of the differential equa- tions of each kinetic model with parameters h. Differential equations were solved by an Adams ⁄ BDF pair as imple- mented in the LSODA routine of odepack [40]. Modelling and computer simulation Mathematical modelling and computer simulation were used to evaluate the relative importance of critical parame- ters of the glyoxalase pathway in L. infantum . Simulations were performed with the software package plas (A. E. N. Ferreira, University of Lisbon, Portugal; http://www.dqb.fc.ul.pt/docentes/aferreira/plas.html) based on a kinetic model of the glyoxalase pathway (Fig. 2) des- cribed in Table 3. In this model, we assumed that the glyoxalase pathway is only dependent on trypanothione and all the variables rep- resent the concentration of total free thiol groups (T(SH) 2 ), total hemithioacetals and total lactoyl-thiol derivatives (SDL-TSH). Bis and mono forms were not differentiated in the model. The response of steady-state concentrations to variations of model parameters (flux of methylglyoxal formation, ini- tial T(SH) 2 concentration and glyoxalase activities) were simulated. Acknowledgements Work supported by project POCTI ⁄ ESP ⁄ 48272 ⁄ 2002 from the Fundac¸ a ˜ o para a Cieˆ ncia e a Tecnologia, Ministe ´ rio da Cieˆ ncia e Tecnologia, Portugal. References 1 Muller S, Liebau E, Walter RD & Krauth-Siegel RL (2003) Thiol-based redox metabolism of protozoan parasites. Trends Parasitol 19, 320–328. 2 Hannaert V, Saavedra E, Duffieux F, Szikora JP, Rig- den DJ, Michels PA & Opperdoes FR (2003) Plant-like traits associated with metabolism of Trypanosoma para- sites. Proc Natl Acad Sci USA 100, 1067–1071. 3 Lohman K & Meyerhof O (1934) U ¨ ber die enzyma- tische umwandlung von phosphoglyzerinsa ¨ ure in brenz- traubensa ¨ ure und phosphorsa ¨ ure (Enzymatic transformation of phosphoglyceric acid into pyruvic and phosphoric acid). Biochem Z 273, 60–72. 4 Richard JP (1984) Acid-base catalysis of the elimination and isomerization reactions of triose phosphates. JAm Chem Soc 106, 4926–4936. 5 Richard JP (1991) Kinetic parameters for the elimin- ation reaction catalyzed by triosephosphate isomerase and an estimation of the reaction’s physiological signifi- cance. Biochemistry 30, 4581–4585. 6 Ferguson GP, Totemeyer S, MacLean MJ & Booth IR (1998) Methylglyoxal production in bacteria: suicide or survival? Arch Microbiol 170, 209–219. 7 Saadat D & Harrison DH (1998) Identification of cata- lytic bases in the active site of Escherichia coli methyl- glyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues. Biochemistry 37, 10074–10086. 8 Cooper RA (1984) Metabolism of methylglyoxal in microorganisms. Annu Rev Microbiol 38, 49–68. 9 Westwood ME & Thornalley PJ (1997) Glycation and advanced glycation endproducts. In The Glycation Hypothesis of Artherosclerosis (Colaco C, ed.), pp. 57–87. Springer-Verlag, Heidelberg. 10 Leoncini G, Maresca M & Bonsignore A (1980) The effect of methylglyoxal on the glycolytic-enzymes. FEBS Lett 117, 17–18. 11 Thornalley PJ (1990) The glyoxalase system: new devel- opments towards functional characterization of a meta- bolic pathway fundamental to biological life. Biochem J 269, 1–11. 12 Darling TN & Blum JJ (1988) d-Lactate production by Leishmania braziliensis through the glyoxalase pathway. Mol Biochem Parasit 28, 121–127. 13 Irsch T & Krauth-Siegel RL (2004) Glyoxalase II of African trypanosomes is trypanothione-dependent. J Biol Chem 279, 22209–22217. 14 Martins AM, Cordeiro CA & Ponces Freire AM (2001) In situ analysis of methylglyoxal metabolism in Saccharo- myces cerevisiae. FEBS Lett 499, 41–44. 15 Mclellan AC, Phillips SA & Thornalley PJ (1992) The assay of methylglyoxal in biological-systems by derivati- zation with 1,2-diamino-4,5-dimethoxybenzene. Anal Biochem 206, 17–23. 16 Bakker BM, Michels PAM, Opperdoes FR & Westerhoff HV (1997) Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes. J Biol Chem 272, 3207–3215. 17 Martins AM, Cordeiro C & Freire AP (1999) Glyoxa- lase II in Saccharomyces cerevisiae: in situ kinetics using the 5,5¢-dithiobis (2-nitrobenzoic acid) assay. Arch Bio- chem Biophys 366, 15–20. 18 Vickers TJ, Greig N & Fairlamb AH (2004) A trypa- nothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major. Proc Natl Acad Sci USA 101, 13186–13191. 19 Iozef R, Rahlfs S, Chang T, Schirmer H & Becker K (2003) Glyoxalase I of the malarial parasite Plasmodium falciparum: evidence for subunit fusion. FEBS Lett 554, 284–288. 20 Martins AM, Mendes P, Cordeiro C & Freire AP (2001) In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae. Eur J Bio- chem 268, 3930–3936. M. Sousa Silva et al. The glyoxalase pathway in Leishmania infantum FEBS Journal 272 (2005) 2388–2398 ª 2005 FEBS 2397 [...]... Synthesis and chromatography of 1,2-diamino-4,5-dimethoxybenzene, 6,7-dimethoxy-2-methylquinoxaline and 6,7-dimethoxy2,3-dimethylquinoxaline for use in a liquid-chromatographic fluorometric assay of methylglyoxal Anal Chim Acta 263, 137–142 2398 M Sousa Silva et al 31 Vander Jagt DL, Han LP & Lehman CH (1972) Kinetic evaluation of substrate specificity in the glyoxalase- Icatalyzed disproportionation of ketoaldehydes... Oza SL, Tetaud E, Ariyanayagam MR, Warnon SS & Fairlamb AH (2002) A single enzyme catalyses formation of trypanothione from glutathione and spermidine in Trypanosoma cruzi J Biol Chem 277, 35853–35861 29 Oza SL, Ariyanayagam MR, Aitcheson N & Fairlamb AH (2003) Properties of trypanothione synthetase from Trypanosoma brucei Mol Biochem Parasitol 131, 25–33 30 Mclellan AC & Thornalley PJ (1992) Synthesis... (2004) Validation of Trypanosoma brucei trypanothione synthetase as drug target Free Radic Biol Med 36, 1289–1302 26 Oza SL, Shaw MP, Wyllie S & Fairlamb AH (2005) Trypanothione biosynthesis in Leishmania major Mol Biochem Parasitol 139, 107–116 27 Comini MA, Guerrero SA, Haile S, Menge U, Lunsdorf H & Flohe L (2004) Validation of Trypanosoma brucei trypanothione synthetase as drug target Free Radic Biol... 3735–3740 32 Castro H, Sousa C, Novais M, Santos M, Budde H, ´ Cordeiro-da-Silva A, Flohe L & Tomas A (2004) Two linked genes of Leishmania infantum encode tryparedoxins localised to cytosol and mitochondrion Mol Biochem Parasit 136, 137–147 33 Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding Anal Biochem 72,.. .The glyoxalase pathway in Leishmania infantum 21 Freire AP, Ferreira A, Gomes R & Cordeiro C (2003) Anti-glycation defences in yeast Biochem Soc Trans 31, 1409–1412 22 Agar NS, Board PG & Bell K (1984) Studies of erythrocyte glyoxalase- II in various domestic species – discovery of glyoxalase- II deficiency in the horse Anim Blood Groups Bi 15, 67–70 23 Karg E, Nemeth I, Horanyi M, Pinter S,... 254 34 Tang D, Shafer MM, Vang K, Karner DA & Armstrong DE (2003) Determination of dissolved thiols using solid-phase extraction and liquid chromatographic determination of fluorescently derivatized thiolic compounds J Chromatogr A 998, 31–40 35 Ondarza RN, Iturbe A, Hurtado G, Tamayo E, Ondarza M & Hernandez E (1999) Entamoeba histolytica: a eukaryote with trypanothione metabolism instead of glutathione... metabolism Biotechnol Appl Biochem 30, 47–52 36 Cordeiro C & Ponces Freire A (1996) Methylglyoxal assay in cells as 2-methylquinoxaline using 1,2-diaminobenzene as derivatizing reagent Anal Biochem 234, 221– 224 37 Thornalley PJ (1988) Modification of the glyoxalase system in human red blood cells by glucose in vitro Biochem J 254, 751–755 38 Abecasis J, Ferreira AE & Ponces Freire A (2004) Metabolic modelling. .. Pinter S, Vecsei L & Hollan S (2000) Diminished blood levels of reduced glutathione and alpha-tocopherol in two triosephosphate isomerase-deficient brothers Blood Cells Mol Dis 26, 91–100 24 Helfert S, Estevez AM, Bakker B, Michels P & Clayton C (2001) Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei Biochem J 357, 117–125 25 Comini MA, Guerrero SA, Haile S, Menge U, Lunsdorf... Metabolic modelling using evolutionary algorithms Eur J Biochem 271 (Suppl 1), 88 39 Price KV (1999) An introduction to differential evolution In New Ideas in Optimization (Corne D, Dorigo M & Glover F, eds), pp 79–108 McGraw-Hill, London 40 Hindmarsh AC (1983) ODEPACK, a Systematized Collection of ODE Solvers in Scientific Computing (Stepleman RS, ed), pp 55–64 North-Holland, Amsterdam FEBS Journal 272 (2005) . Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation Marta Sousa Silva 1 ,. evaluating the relevance of the glyoxalase pathway as a potential therapeutic target by revealing the importance of critical parameters of this pathway in