Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates.. The sensitivity
Trang 1Leishmania infantum as a therapeutic target by modelling and computer simulation
Marta Sousa Silva1, Anto´nio E N Ferreira1, Ana Maria Toma´s2,3, Carlos Cordeiro1
and Ana Ponces Freire1
1 Centro de Quı´mica e Bioquı´mica, Departmento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias da Universidade de Lisboa, Portugal
2 ICBAS – Instituto de Cieˆncias 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 N1,N8
-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 Cieˆncias
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
Km of 0.253 mm and a V of 0.21 lmolÆmin)1Æmg)1 for glyoxalase I, and a
Km of 0.098 mm and a V of 0.18 lmolÆmin)1Æmg)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-tumis 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.
Trang 2conditions, 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; lactoylS-d-lactoyl-glutathione
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 108 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 abspres-ence 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
C 3 O
O H
C 3 OH
O S R
H
C
3
OH
O OH H
D-Lactate
Glutathione or trypanothione -SH group Hemithioacetal
Methylglyoxal
Dihydroxyacetone
phosphate
3-P-1,2-enediol
D-glyceraldehyde -3-phosphate
O3POCH2
O
H
OH
H
H O
O3POCH2 OH
O3POCH2
OH
O H H
(non-enzymatic)
(non-enzymatic)
2-C
3
S R O
O
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 ].
Trang 3and 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 108 promastigotes, while GSH
concen-tration was 0.50 nmol per 108 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 Kmof 1.85 ± 0.35 mm and a V of
0.19 ± 0.02 lmolÆmin)1Æmg)1 (Table 1) The Km 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 Km of 0.24 ± 0.04 mm and a
V of 0.19 ± 0.02 lmolÆmin)1Æmg)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.
Trang 4Glx II, the activity could be measured and we obtained
a Km of 0.073 ± 0.020 mm and a V of 0.22 ±
0.0005 lmolÆmin)1Æmg)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
Km of 0.253 mm and an apparent V of 0.21 lmolÆ min)1Æmg)1 (Table 1) while for Glx II a Km of 0.098 mm and a V of 0.18 lmolÆmin)1Æmg)1were 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 Km of 0.801 mm and a V of 0.5 lmolÆmin)1Æmg)1were deter-mined, markedly different from the ones estimated from initial rate and time course analysis using the two-enzyme model Moreover, the obtained Ki 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.
K m
(m M )
V (lmolÆmin)1Æmg)1)
K m
(m M )
V (lmolÆmin)1Æmg)1)
a
NC, not comparable (data from recombinant enzyme).bNC, not comparable (data from permeabilized cells).
Table 2 Glyoxalase II kinetic parameters in Leishmania infantum and other cells ND, not detected.
Km (m M )
V (lmolÆmin)1Æmg)1)
Km (m M )
V (lmolÆmin)1Æmg)1)
a NC, not comparable (data from recombinant enzyme) b NC, not comparable (data from permeabilized cells).
Trang 5possible 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 concentraforma-tion
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
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¢.
Trang 6possible 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
D C
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 inpu t
initia
l SH
0 20 60
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
initia
l SH
0 20 60
0 20 40 60 80
0.2 0.4 0.6 0.8 1.0 MG
GLX I
GLX II
0 20 60
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 60
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).
Trang 7pathway, 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 concentraforma-tion 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
DHAP, methylglyoxal dimethylacetal, trifluoroacetic acid
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 DU (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
Preparation of metabolites
High-purity MG was prepared by acid hydrolysis of
Trang 8by 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
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
[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
MA67ITMAP263 were grown in RPMI medium
supple-mented with 10% fetal bovine serum, 2 mm l-glutamine,
Preparation of Leishmania infantum extracts
Promastigotes of L infantum at early stationary phase of
cells were submitted to eight freeze–thaw cycles (on ice and
centrifuga-tion at 10 500 g for 10 min Protein concentracentrifuga-tion 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
[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
centrifuged at room temperature for 3 min at 10 500 g The
reduction of oxidized thiols was performed with
aceto-nitrile) was added to a final concentration of 1 mm (200 lL reaction system) and the derivatization was carried out at
concentration of 0.5 m, was added to stop the reaction
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
TFA (solvent B) The gradient program was: 0–5 min, 10%
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
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
occurred in 1 mL reaction volume, in 0.1 m potassium
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
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
MG in excess (3.34 mm) Initial concentrations of GSH and GSSG were calculated to give hemithioacetal
Trang 9were 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 reacaddi-tions 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
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
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]
HTA
1
1 1
+
=
m
K
V v
In the ‘two-enzyme model’, the consecutive reactions of
gly-oxalase I and glygly-oxalase II, both with irreversible Michaelis–
Menten rate laws were considered (Scheme 2)
[HTA]
HTA
1
1
1
+
=
m
K
V
[SDL - TSH]
TSH -SDL 2
2 2 +
= m
K
V v
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)
[SDL-TSH] [ ]HTA 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):
k¼1
i¼1
k ðtihÞ
In this equation, p is the number of time courses used in
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 + v2) – v3+ v4 dHTA ⁄ dt ¼ v 3 – v4– v5 dSDLTSH ⁄ dt ¼ v 5 – v 6
dT(SH)2⁄ dt ¼ – v 3 + v4+ v6 Rate equations
v 1 ¼ k 1 GAP
v2¼ k 2 DHAP
v3¼ k 3 MG T(SH)2
v 4 ¼ k 4 HTA
v 5 ¼ V 5 HTA ⁄ (K m5 + HTA)
v6¼ V 6 SDLTSH ⁄ (K m6 + SDLTSH) Parameters
k 1 ¼ 6.4 · 10)3min)1
k2¼ 6.6 · 10)4min)1
k3¼ 0.34 m M )1Æmin)1
k 4 ¼ 1.01 min)1
V5¼ 2 · 3.042 m M Æmin)1
V6¼ 2 · 2.653 m M Æmin)1
K m5 ¼ 2 · 0.253 m M
Km6¼ 2 · 0.0980 m M
GAP ¼ 0.0072 m M
DHAP ¼ 0.16 m M
T(SH) 2 (at time zero) ¼ 2 x 0.45 m M
Trang 10course k at time ti, and XSIM
predicted by the numerical solution of the differential
equa-tions of each kinetic model with parameters h Differential
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
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
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,
simulated
Acknowledgements
Work supported by project POCTI⁄ ESP ⁄ 48272 ⁄ 2002
from the Fundac¸a˜o para a Cieˆncia e a Tecnologia,
Ministe´rio da Cieˆncia 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 J Am
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
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
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)
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
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