Dissecting the catalytic mechanism of trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modeling

Article abstract of DOI:10.1074/jbc.M113.483289

Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form trypanothione. Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing the entire kinetic profile. Conclusion: Trypanothione synthetase is affected by substrate and product inhibition. Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this parasite-specific enzyme.

Full text of DOI:10.1074/jbc.M113.483289

Enzymology:
Dissecting the Catalytic Mechanism of
Trypanosoma brucei Trypanothione
Synthetase by Kinetic Analysis and
Computational Modeling
Alejandro E. Leroux, Jurgen R. Haanstra,
Barbara M. Bakker and R. Luise
Krauth-Siegel
J. Biol. Chem. 2013, 288:23751-23764.
doi: 10.1074/jbc.M113.483289 originally published online June 28, 2013
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 33, pp. 23751–23764, August 16, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Author’s Choice
Dissecting the Catalytic Mechanism of Trypanosoma brucei
Trypanothione Synthetase by Kinetic Analysis and
*
Computational Modeling
Received for publication, May 15, 2013, and in revised form, June 26, 2013 Published, JBC Papers in Press, June 28, 2013, DOI 10.1074/jbc.M113.483289
‡
1
§¶1
§¶2
‡3
Alejandro E. Leroux , Jurgen R. Haanstra , Barbara M. Bakker , and R. Luise Krauth-Siegel
‡
§
From the Biochemie-Zentrum der Universität Heidelberg, D-69120 Heidelberg, Germany, University of Groningen, University
Medical Center Groningen, Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Groningen NL-9713 GZ,
The Netherlands, and the Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University, Amsterdam
¶
NL-1081 HV, The Netherlands
Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form
trypanothione.
Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing
the entire kinetic profile.
Conclusion: Trypanothione synthetase is affected by substrate and product inhibition.
Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this
parasite-specific enzyme.
In pathogenic trypanosomes, trypanothione synthetase (TryS) diate Gsp dissociates from the enzyme between the two catalytic
catalyzes the synthesis of both glutathionylspermidine (Gsp) and steps, and (iii) T(SH) inhibits the enzyme by remaining bound
2
trypanothione (bis(glutathionyl)spermidine (T(SH) )). Here we at its product site and, as does the inhibitory GSH, by binding to
2
present a thorough kinetic analysis of Trypanosoma brucei the activated enzyme complex. The newly detected concerted
TryS in a newly developed phosphate buffer system at pH 7.0 substrate and product inhibition suggests that TryS activity is
and 37 °C, mimicking the physiological environment of the tightly regulated.
enzyme in the cytosol of bloodstream parasites. Under these
conditions, TryS displays K values for GSH, ATP, spermidine,
m
and Gsp of 34, 18, 687, and 32 ␮M, respectively, as well as K
Trypanosoma and Leishmania are the causative agents of
i
values for GSH and T(SH) of 1 mM and 360 ␮M, respectively. As tropical diseases such as African sleeping sickness and Nagana
2
Gsp hydrolysis has a K value of 5.6 mM, the in vivo amidase cattle disease (Trypanosoma brucei species), South-American
m
activity is probably negligible. To obtain deeper insight in the Chagas disease (Trypanosoma cruzi), and the different forms of
molecular mechanism of TryS, we have formulated alternative leishmaniasis. A common feature of these parasitic protozoa is
kinetic models, with elementary reaction steps represented by their unusual thiol redox metabolism. They lack glutathione
linear kinetic equations. The model parameters were fitted to reductases and thioredoxin reductases but have a trypanothione/
the extensive matrix of steady-state data obtained for different trypanothione reductase-based system instead. Trypanothione
1
8
4
substrate/product combinations under the in vivo-like condi- (N ,N -bis(glutathionyl)spermidine(T(SH) ) )isofpivotalimpor-
2
tions. The best model describes the full kinetic profile and is able tancefortheviabilityandvirulenceoftheseparasites. Thedithiolis
to predict time course data that were not used for fitting. This the donor of reducing equivalents for the synthesis of DNA pre-
system’s biology approach to enzyme kinetics led us to conclude cursors by ribonucleotide reductase and the detoxification of
that (i) TryS follows a ter-reactant mechanism, (ii) the interme- hydrogen peroxide and lipid-derived hydroperoxides by different
tryparedoxin peroxidases as well as the reduction of protein
methionine sulfoxides (for a recent review, see Ref. 1).
*
This work was supported by the Bundesministerium für Bildung und Forsc-
hung (BMBF), Germany, and the Netherlands Organization for Scientific
T(SH) is synthesized from two molecules of GSH that are
2
Research (NWO), The Netherlands, within the framework of the SysMo2- covalently bridged by a spermidine (Spd) in two ATP-depen-
funded SilicoTryp project of the European Union (work packages 4 (to dent reactions. In the first step, glutathionylspermidine (Gsp) is
B. M. B.) and 6 (to R. L. K.-S.).
formed, which is then combined with the second GSH to yield
Author’s Choice—Final version full access.
Both authors contributed equally.
1
2
T(SH) . In Crithidia fasciculata, an insect parasite widely stud-
2
Recipient of a Rosalind Franklin Fellowship from the University of Gronin- ied as model trypanosomatid, two enzymes, a glutathionylsper-
gen. Towhomcorrespondencemaybeaddressed:Dept. ofPediatrics, Cen-
midine synthetase (GspS) and trypanothione synthetase (TryS),
ter for Liver, Digestive, and Metabolic Diseases, University of Groningen,
University Medical Centre Groningen CA84, Hanzeplein 1, NL-9713 GZ
are present (2, 3). A GspS, but not TryS, also occurs in Esche-
EG - rmo na ii nl : gb e. mn ,. bT ah k ek eNr 0e 1t h@ e ur lma nc dg s. n. l T. el.: 31-50-3080407; Fax: 31-50-361-1746; richia coli, the first organism for which a direct linkage of the
To whom correspondence may be addressed: Biochemie-Zentrum der Uni-
3
4
versität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg,
Germany. Tel.: 49-6221-544187; Fax: 49-6221-545586; E-mail: luise.krauth-
siegel@bzh.uni-heidelberg.de.
The abbreviations used are: T(SH) , trypanothione; TryS, trypanothione syn-
2
thetase; Spd, spermidine; Gsp, glutathionylspermidine; GspS, glutathio-
nylspermidine synthetase.
AUGUST 16, 2013•VOLUME 288•NUMBER 33
JOURNAL OF BIOLOGICAL CHEMISTRY 23751

Catalytic Mechanism of Trypanothione Synthetase
based on knowledge of the kinetic parameters for all enzymes
under identical conditions (21). Even more importantly, the
kinetic data should be measured under conditions that resem-
ble the milieu in which the pathway is active rather than at a
non-physiological optimum (22). The importance of intracel-
lular conditions for metabolic functions could recently be dem-
onstrated; that is, the use of an “in vivo-like” assay medium
yielded enzyme kinetic parameters that substantially improved
a computational model of yeast glycolysis (23). Such data are
not yet available for the parasite T(SH) metabolism. As in
2
other cases, the proteins were characterized under conditions
giving the highest activities for the individual enzyme studied.
Thus, different buffer compositions, ionic strengths, and pH
values were applied. For instance, the kinetic parameters of
T. brucei TryS were measured in HEPPS buffer at pH 8.0 and
2
5 °C (7). Another characteristic of the intracellular milieu is
the simultaneous presence of substrates and products, but
product inhibition of TryS has not been investigated either.
Here we present a comprehensive analysis of T. brucei TryS
under conditions that resemble the in vivo situation. We devel-
oped a phosphate-based buffer system at pH 7.0 that mimics
the cytosol of bloodstream African trypanosome and is recom-
mended for the comparative studies of all enzymes from the
T. brucei cytosol. The complexity of the catalytic mechanism of
TryS, including two catalytic cycles and inhibition by the sub-
SCHEME 1
GSH and Spd metabolism was described (4, 5). Gsp is formed strate GSH as well as by the product T(SH) , led us to build a
2
when the bacteria enter the stationary phase and is broken mathematical model that allowed the incorporation of all
down as soon as growth is restored. Both reactions are catalyzed kinetic data. The combination of measurements under in vivo-
by GspS, which is a bifunctional enzyme with Gsp synthetase like conditions at 37 °C with computational modeling indeed
and amidase activity (6).
provided us with interesting novel insights in the kinetic mech-
In contrast to C. fasciculata, the pathogenic T. brucei (7, 8), anism of TryS.
T. cruzi (9, 10), and Leishmania major (11) species employ TryS
EXPERIMENTAL PROCEDURES
as a single enzyme to generate both Gsp and T(SH) (Scheme 1).
2
In vitro, TryS also displays an amidase activity and in total can
Materials—NADH was purchased from Biomol, monobro-
catalyze five different reactions: ATP hydrolysis and formation mobimane was from Calbiochem, HEPES and Tris were from
of Gsp and T(SH) as well as hydrolysis of the conjugates to Roth, and Spd, GSH, ATP, phosphoenolpyruvate, rabbit pyru-
2
regenerate GSH and Spd. The related GspS enzymes from vate kinase, and bovine L-lactate dehydrogenase were from
E. coli and C. fasciculata catalyze the latter reaction at pH 7.5 Sigma. T(SH) and trypanothione disulfide (TS ) were pre-
2
2
and 25 °C, with a K value of 0.9 and 0.5 mM and a k of 2.1 and pared enzymatically (24). Gsp was produced essentially by the
m
cat
Ϫ1
5
0
.38 s , respectively (2, 6). The amidase activity of the TryS same procedure, replacing TryS by C. fasciculata C79A GspS.
enzymes appears to be comparably low. T. cruzi and T. brucei
Cloning, Overexpression, and Purification of Recombinant
TryS hydrolyze Gsp at a rate that is less than 1% that of the Tag-free TryS—The coding region of TryS was amplified from
synthetase activity, and cleavage of T(SH) is even slower (7, 9). genomic DNA of the T. brucei 449 strain using Pfu DNA
2
In the search for novel drug targets, pathways that are essen- polymerase (Fermentas) (94 °C for 2 min, 94 °C for 30 s, 57 °C
tial for parasite survival and are absent from the mammalian for 30 s, and 72 °C for 2 min for 25 cycles and 72 °C for 10 min).
host are attractive starting points. Genetic and chemical The forward primer (5Ј-CC ATG GGC ATG ACG AAG TCG-
approaches revealed that all proteins of the T(SH) system 3Ј) contained an NcoI restriction site and the reverse primer
2
studied so far, such as ␥-glutamylcysteine synthetase (12), TryS (5Ј-GG TAC CTA CAT TTG AAT ACG TAC GGG A-3Ј)
(
8, 13), trypanothione reductase (14), and tryparedoxin (15) as introduced an Acc65I site after the stop codon. The PCR prod-
well as the cytosolic 2-Cys-peroxiredoxin (16) and glutathione uct was ligated into the pET-blue1 vector (Novagen) and ampli-
peroxidase-type enzymes (16–18) are essential for the viability fied in E. coli Nova Blue cells (Novagen). An NcoI site in the
of African trypanosomes. Several of them are currently investi- coding region was removed with the QuikChange௡ site-di-
gated as potential drug targets (19). From both a metabolic rected mutagenesis kit (Stratagene) with the primers TryS-
point of view and putative druggability, TryS has been sug- mut-F (GTT AAT GAG GAT GCG CCG TGG GGA CAT GTC
gested to have the highest therapeutic potential of all enzymes GCG) and TryS-mut-R (CGC GAC ATG TCC CCA CGG CGC
of the T(SH) system (19, 20).
ATC CTC ATT AAC) following the provider’s instructions.
2
A final conclusion as to which protein plays a main role in the
5
pathway control would need a reliable computational model
K. Diederich, C. Hiller, and R. L. Krauth-Siegel, unpublished work.
2
3752 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 33•AUGUST 16, 2013

Catalytic Mechanism of Trypanothione Synthetase
After digestion with NcoI and Acc65I, the fragment was ligated
into the pETtrx1b vector (kindly provided by Gunther Stier)
and fully sequenced (GATC Biotech AG, Konstanz, Germany).
E. coli Tuner(DE3) competent cells (Novagen) were trans-
formed with the plasmid and grown in 1 liter of Terrific Broth
medium containing 50 ␮g/ml kanamycin at 37 °C. At an OD
6
00
of 0.6 the expression was induced by 0.3 mM isopropyl-␤-D-
thiogalactopyranoside. The cells were allowed to grow over-
night at 25 °C, harvested, and resuspended in 30 ml of buffer A
(
50 mM Tris-HCl, 300 mM NaCl, pH 8.0) containing 150 nM
pepstatin, 4 nM cystatin, 100 ␮M phenylmethylsulfonyl fluoride,
6
mg of lysozyme, and 0.6 mg of DNase A. The bacteria were
disintegrated by sonication, and the clarified extract was loaded
onto a nickel-nitrilotriacetic acid Superflow matrix (Qiagen)
following an established procedure (25). The fusion protein was
eluted with 30 mM imidazole in buffer A, concentrated on a
5
4
0-kDa Amicon filter (Millipore), and digested overnight at
°C with His-tagged tobacco etch virus protease (26). About 10
mg of tag-free TryS was collected from the run-through of a
second nickel column. The purity of the protein was Ն95%
according to SDS-PAGE. Recombinant TryS was stored at a
concentration of 2 mg/ml at 4 °C. The protein concentration
was determined by a Bradford assay using bovine serum albu-
min as standard. A 1 mg/ml solution of pure tag-free T. brucei
TryS shows an A
of 1.9.
2
80 nm
Kinetic Characterization of the Synthetase Activity—To allow
comparison with published data (7), TryS was first studied at
2
5 °C and 37 °C in 100 mM K-HEPES, pH 8.0, supplemented
with 5 mM dithiothreitol, 0.5 mM EDTA, and 10 mM MgCl . To
2
mimic the physiological environment of the enzyme, the activ-
ity was measured at 37 °C in 10 mM potassium phosphate, pH
7
.0, containing with 15 mM NaCl, 85 mM KCl, and 10 mM MgCl
2
(
in vivo-like buffer system). The substrate stock solutions were
prepared in the respective buffer and, if necessary, the pH was
adjusted. The ATP stock solution consisted each of 100 mM
ATP and MgCl . The assays were performed in a final volume of
2
0
0
0
.2 ml containing 0.2 mM NADH, 1 mM phosphoenolpyruvate,
.4 units of pyruvate kinase, 0.4 units of lactate dehydrogenase,
.2–0.8 ␮M TryS, and varying concentrations of ATP, GSH, and
Spd or Gsp. The standard assay contained ATP, GSH, and Spd
at fixed concentrations of 2.1, 0.1, and 20 mM, respectively. The FIGURE 1. Steady-state kinetic analysis of TryS. The activities were meas-
ured in the in vivo-like buffer system varying the concentration of two sub-
strates while keeping one constant at saturating concentrations. Shown are
double reciprocal plots of the reactions with fixed concentrations: 8 mM Spd
assay mixtures were preincubated for 2 min before the reaction
was started by the addition of Spd or Gsp and the absorption
decrease at 340 nm was followed in a UV-visible V-650 spectro- (A), 2.3 mM ATP (B), and 2.3 mM ATP (C).
photometer (Jasco). After each assay under the in vivo-like con-
ditions, the pH of the reaction mixture was controlled to
To get the highest possible data density, we have chosen not
to replicate each individual experiment but rather measure at
exclude significant changes. The apparent K values and k
m
cat
were determined by varying the respective substrate at fixed many different substrate and product concentrations. There-
concentrations of the co-substrates (2.3 mM ATP, 1.0 mM GSH, fore, Figs. 1–3 show single measurements. The full dataset con-
and 8 mM Spd, or 0.5 mM Gsp). The concentrations ranged sists of 288 single measurements and is available at SysMO
between 0.005 and 2.5 mM for ATP, 0.04 and 2.0 mM for GSH, SEEK database. The internal consistency of the dataset is dem-
0
.12 and 8.0 mM for Spd, and 0.01 and 0.50 mM for Gsp. The onstrated by the smoothness of curves and the reproducible
parameters were determined by non-linear regression; the results obtained for occasional replicates.
experimental data were fitted to the specific function applying
HPLC Quantification of Gsp and T(SH) —In 2 ml of the in
2
the Gauss-Newton algorithm (27). All data used for kinetic vivo-like buffer, 170 nM TryS was mixed with 2.1 mM ATP and
model construction were obtained under the in vivo-like con- different concentrations of GSH and Spd as well as all compo-
ditions. Product inhibition of TryS was followed by varying one nents for the coupled enzymatic assay (see above), and NADH
substrate at fixed concentrations of T(SH) .
consumption was followed at 37 °C in the spectrophotometer.
2
AUGUST 16, 2013•VOLUME 288•NUMBER 33
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Catalytic Mechanism of Trypanothione Synthetase
At different time points (during the first 2 min every 20 s and ice-cold trichloroacetic acid. After centrifugation, the superna-
then at 4, 8, 15, and 24 min), a 100-␮l aliquot was removed, and tant was extracted with diethyl ether. The thiols were deriva-
the reaction was immediately stopped by adding 10 ␮l of 100% tized with monobromobimane, and the samples were subjected
to HPLC analysis as previously described (28). Standard solu-
tions containing 5, 20, or 50 ␮M of each Gsp and T(SH) were
2
prepared under identical conditions but omitting TryS.
ATPase and Amidase Activity of TryS—The ATPase activity
was assessed using the synthetase assay lacking at least one of
the other two substrates. The assays contained between 0.2 and
1
0 mM ATP and 2–10 ␮M TryS. The amidase activity was meas-
ured at 37 °C in 0.6 ml of the in vivo-like buffer containing 1.2
M TryS and 0.1, 0.5, 2, or 8 mM Gsp. After 2, 5, 10, 30, and 60
␮
min, a 100-␮l aliquot was removed, and the reaction immedi-
ately stopped by adding 10 ␮l of 100% ice-cold trichloroacetic
acid. The samples were diluted with 10% trichloroacetic acid to
a maximum thiol concentration of 0.4 mM. The thiols were
derivatized and analyzed by HPLC as described above. The ini-
tial velocities were calculated from the GSH produced within 5
and 10 min, respectively. To evaluate the effect of ATP on the
amidase activity, reaction mixtures contained 1.2 ␮M TryS, 0.5
mM Gsp, and 0, 0.4, and 2.1 mM ATP, respectively.
Kinetic Model and Simulations—PySCeS (Python Simulator
for Cellular Systems) open source modeling software (29) was
used for initial model construction. Each elementary reaction
step was described by reversible mass action kinetics, except the
phosphorylation steps of GSH, which were modeled as irrevers-
ible. Alternative model versions were exported from PySCeS in
sbml format to allow import into COPASI version 4.49.45 (30)
for parameter estimation as well as steady-state and time course
simulations. Constraints on the parameters were added to
enforce microscopic reversibility in the central part of the
FIGURE 2. Substrate inhibition of TryS by GSH. The activity of TryS at vari-
able concentrations of GSH was measured in the in vivo-like buffer system.
The assays contained 8 mM Spd and different fixed ATP concentrations (A)
and 2.3 mM ATP and different fixed Spd concentrations (B). Data were fitted to
the high substrate inhibition equation as described under “Results.”
FIGURE 3. Product inhibition of TryS by T(SH) . The assays were conducted in the in vivo-like buffer system. The reaction mixtures contained in the presence
2
or absence of T(SH) 0.2 mM GSH, 2.3 mM ATP, and variable Spd concentrations (A), 0.2 mM GSH, 2.3 mM ATP, and variable Gsp concentrations (B), 8 mM Spd, 2.3
2
mM ATP, and variable GSH concentrations (C), and 0.52 mM Gsp, 2.3 mM ATP, and variable GSH concentrations (D). Data in A and B were fitted to the
Michaelis-Mentenequation. Thekineticconstantsderivedaregivenbelowthegraphs. DatainCandDwerefittedtothehighsubstrateinhibitionequation(solid
lines) and for the highest T(SH) concentration also to the Michaelis-Menten equation (dashed line).
2
2
3754 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 33•AUGUST 16, 2013

Catalytic Mechanism of Trypanothione Synthetase
model. For each face of the cubes (i.e. E-EA-EAB-EB-E) (Fig. 6) were never included in the parameter estimations but were
the product of the rate constants going clockwise should equal used for independent validation.
the product of the rate constants going counterclockwise
RESULTS
(
31). The concentrations in the model are in ␮M, and the time
unit is in seconds. Fluxes of individual reactions within the
Setup of a Buffer System That Mimics the Cytosol of Blood-
model are in ␮M/s. For Spd as substrate, steady-state calcula- stream T. brucei—Based on published data (32–34), a phos-
tions were done at fixed concentrations of ATP, GSH, Spd, and phate buffer system was established which mimics the cytosol
of the cell and is composed of 100 mM potassium (32), 15 mM
sodium (32), 10 mM magnesium, 120 mM chloride (32), 10 mM
total phosphate and has a pH of 7.0 (33, 34). From the total
magnesium content of T. brucei (35), a concentration of 6 mM
magnesium in the cytosol of bloodstream cells could be esti-
mated. This value fits to the 0.3–1.5 mM free magnesium
reported for other cells taking into account that the total con-
centration is 10–20 times higher (36). In addition, polyamines
can compete with magnesium for ATP binding (36). Thus, a
magnesium concentration of 10 mM was selected, and the ATP
stock solution was made of each 100 mM concentration of ATP
T(SH) . For Gsp, the procedure was the same except that the
2
Gsp concentration was also fixed and that of Spd was zero. The
model contains an irreversible reaction that consumes ADP,
which mimics the coupling reaction in the enzymatic assay. In
steady state, the rate of this reaction is equivalent to the overall
ADP formation flux. For the steady-state simulations the total
enzyme concentration, which is a conserved moiety, was set to
1
␮M, and the calculated rates were normalized to enzyme con-
centration. Therefore, the overall ADP formation flux by the
model is expressed in ␮M/s per 1 ␮M enzyme, leading to a unit of
Ϫ1
s
. The time courses were simulated at 0.17 ␮M TryS, which
and MgCl to ensure the presence of Mg-ATP throughout the
2
was the enzyme concentration used in the corresponding
ATP concentration range studied.
experiment, with Gsp and T(SH) as variables.
2
The total concentration of inorganic phosphate in T. brucei
has been found to be 20–40 mM (37). The cytosolic level may be
considerably lower because of the high phosphate concentra-
tion in the acidocalcisomes of the parasites. The activity of TryS
was about 25% diminished in a 30 mM phosphate compared
with 10 mM phosphate buffer, pH 7.0. This was not due to the
higher ionic strength because the addition of 100 mM NaCl to
the HEPES buffer, pH 8.0, did not affect the enzyme activity
Parameter Estimation—The kinetic constants in the model
were fitted to the steady-state data obtained in the in vivo-like
buffer. Parameter estimation was done in COPASI (30) by evo-
lutionary programming (200 generations, population size of
2
0). The rate of the coupling reaction (which in steady state
equals the ADP formation flux) was compared with the meas-
Ϫ1
ured ADP production rate (in s ), and the least sum of squares
was the objective function to be minimized, E(P) ϭ ⌺ ␻ ϫ
i,j
j
(data not shown). As different mammalian cells have been
2
(
x Ϫ y (P)) , in which E is the objective value, P is the tested
i,j
i,j
reported to have cytosolic phosphate concentrations between 1
and 10 mM (38–40), the phosphate concentration was finally
set to 10 mM. The overall calculated ionic strength of this in
vivo-like buffer is 200 mM, which corresponds to that in red
blood cells (41). Macromolecular crowding agents (polyethyl-
ene glycol or BSA) mimicking the high intracellular protein
concentration did not affect the enzyme activity significantly
and were therefore not included in the assay medium.
parameter set, x is a point in the dataset, and y (P) is the
i,j
i,j
corresponding simulated value. The indices i and j denote rows
and columns in the dataset. The weight for each data column is
given by ␻ , which was set to a fixed value of one in all our
j
calculations.
Parameter values to be fitted were allowed to vary between
Ϫ10
1
ϫ 10
and infinity. Because the Gsp concentration was a
fixed parameter when it was the substrate but a variable inter-
mediate with Spd as substrate, we needed subsequent rounds of
parameter estimations using alternately the data on Spd or Gsp.
The parameter estimation was started with a value of 100 for
each parameter, and the Gsp data were fitted allowing all
parameters to vary. Continuing from the resulting parameter
set, the Spd data were fitted varying only the parameters of the
Spd branch (see Fig. 6) with Gsp concentration as a variable.
Next, the parameters of the Gsp branch were refitted to the Gsp
data, and in a final round the parameters of the Spd branch were
refitted to the Spd data. Additional rounds did not decrease the
overall objective value (sum of least squares on Spd data and
Gsp together).
Buffer Species, pH, and Temperature Strongly Affect the
Kinetic Constants of TryS—TryS was analyzed in HEPES buffer
at pH 8.0 and 7.0 and in the in vivo-like phosphate buffer, pH
7
.0. Assays were conducted at 25 °C to allow comparison with
published data and at 37 °C, reflecting the physiological envi-
ronment of the parasite in the mammalian host. Hyperbolic
kinetics were obtained when ATP, Spd, or Gsp was the variable
substrate. In contrast, TryS displayed substrate inhibition when
GSH was varied. In the latter case, the experimental data were
fitted to Haldane’s high substrate inhibition equation, v ϭ
V
/(1 ϩ K /S ϩ S/K ), which is based on unproductive bind-
max
m
i
ing of GSH to the substituted enzyme (42). This yielded the K
m
and K values for GSH as well as the k depicted in Table 1. At
i
cat
Evaluation of Models—Alternative models with fitted varying ATP, Spd, or Gsp concentrations, the enzyme showed
parameters were evaluated first by comparing the objective pronounced inhibition at GSH concentrations of Ն300 ␮M (see
value of the estimation between the different models. Models below and Fig. 2). Under these conditions, the calculated k
cat
with the lowest objective value were further inspected by visual values were, therefore, lower than those given in Table 1 (see for
comparison of the model calculations with the experimental instance Table 2).
data for key behavior of the enzyme (i.e. how the model per-
The kinetic parameters of TryS obtained in HEPES, pH 8.0, at
forms at different substrates ratios, the shape of the GSH inhi- 25 °C were comparable with published data using HEPPS, pH
bition curve, and inhibition by T(SH) ). The time-course data 8.0 (7). The most obvious difference was the ratio between the
2
AUGUST 16, 2013•VOLUME 288•NUMBER 33
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Catalytic Mechanism of Trypanothione Synthetase
TABLE 1
Spd are 8.30, 9.90, and 10.66 (44), and lowering of the pH affects
Kinetic parameters of T. brucei TryS in different assay systems
their ionization state. The higher K value at pH 7.0 compared
m
The fixed substrate concentrations used in the HEPES system were 2.5 mM ATP, 1
with pH 8.0 suggests that TryS preferably interacts with a less
mM GSH, and 20 mM Spd and in the in vivo-like phosphate buffer 2.5 mM ATP, 2 mM
GSH, 8 mM Spd, and 0.5 mM Gsp. Inhibition of TryS by T(SH) versus Spd or Gsp protonated form of Spd. A similar pH dependence has been
2
was studied at fixed 0.2 mM GSH (for details see the legend of Fig. 3). The kinetic
described for E. coli GspS where changing the pH from 7.5 to
parameters, which are the means of at least two series of experiments Ϯ S.E. were
6
.8 resulted in a 3-fold increase of the K value for Spd (6). With
obtained as described under “Experimental Procedures.” ND, not determined.
m
Ϫ1
HEPPS,
Phosphate,
Gsp as the third substrate, TryS showed a k of 2.1 s , which
cat
a
pH 8.0
HEPES, pH 8.0
pH 7.0
is slightly lower than with Spd. Although not as pronounced as
Kinetic parameters
25 °C
25 °C
37 °C
37 °C
␮M
in the case of Spd, the K value of Gsp also increased with
m
␮
M
␮M
decreasing pH. Taken together, pH, temperature, and buffer
composition have a strong impact on the kinetic parameters of
TryS.
app
K
Spd
Gsp
38 Ϯ 5
92 Ϯ 25
139 Ϯ 14 687 Ϯ 90
m
app
K
2.4 Ϯ 0.2 12 Ϯ 2
16 Ϯ 3
32 Ϯ 5
m
With Spd as substrate
app
K
K
K
k
GSH
GSH
56 Ϯ 11
37 Ϯ 7
32 Ϯ 4
45 Ϯ 3
69 Ϯ 5
Evaluation of the Catalytic Mechanism of TryS by Steady-
state Kinetics—Using the in vivo-like buffer system, a matrix of
kinetic data at various substrate and product concentrations
was collected. At fixed Spd and variable GSH and ATP, the
regression lines in the double reciprocal plot converged, sug-
gesting the presence of a quaternary enzyme-substrate complex
(Fig. 1A). In contrast, at fixed ATP and variable GSH and Spd or
Gsp concentrations, the reciprocal plots revealed a reproduci-
ble but not clear picture (Fig. 1, B and C). The lines did not cut
in a common point, and several of them appeared to be parallel,
a pattern expected for a ping-pong mechanism. However, this is
clearly not the case. In all three series of kinetics, independent
of the nature of fixed and variable substrates, the lines did not
follow the order expected when stepwise changing the GSH
concentration. The activity of TryS increased from 40 to 100
and 300 ␮M GSH but then dropped when the concentration was
m
app
143 Ϯ 20 332 Ϯ 22 849 Ϯ 115
i
app
ATP
7.1 Ϯ 0.4 6.6 Ϯ 0.5 10 Ϯ 2
18 Ϯ 4
m
Ϫ1
(s
)
2.9 Ϯ 0.4 1.8 Ϯ 0.1 5.2 Ϯ 0.2 2.8 Ϯ 0.1
cat
app
b
c
K
T(SH)
T(SH)
ND
ND
ND
ND
ND
ND
228 Ϯ 112
377 Ϯ 64
i
2
2
app
K
i
With Gsp as substrate
app
K
K
K
k
GSH
GSH
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
34 Ϯ 12
m
app
1085 Ϯ 309
12 Ϯ 3
i
app
ATP
m
Ϫ1
(s
)
2.1 Ϯ 0.3
223 Ϯ 100
346 Ϯ 25
cat
app
d
c
K
T(SH)
T(SH)
i
2
2
app
K
i
a
Data are from Oza et al. (7), with 2 mM ATP, 0.1 mM GSH, and 10 mM Spd as
fixed substrate concentrations.
b
c
d
Apparent uncompetitive inhibitor constant versus the substrate Spd.
Apparent uncompetitive inhibitor constant versus the substrate GSH.
Apparent uncompetitive inhibitor constant versus the substrate Gsp.
TABLE 2
Effect of buffer species, pH, and temperature on the kinetic parame-
ters of TryS for Spd
The apparent Km values for Spd were determined at a fixed ATP and GSH concen- further raised (Fig. 1) because GSH is not only a substrate but
tration of 2.1 and 1 mM, respectively. The kinetic parameters, which are the means
also an inhibitor of the enzyme (Table 1, see also the next sec-
of at least two determinations Ϯ S.E., were obtained as described under “Experi-
mental Procedures.”
tion). The irregular pattern when Spd or Gsp was varied (Fig. 1,
B and C) is most likely also due to the GSH inhibitory effect.
Mechanism of Inhibition of TryS by Its Substrate GSH—GSH
acts as both substrate and inhibitor of TryS from several species
app
app
cat
Buffer conditions
K
k
m
Ϫ1
(
␮M)
(s
)
HEPES, pH 8.0 (25 °C)
92 Ϯ 25
0.13 Ϯ 0.01
0.15 Ϯ 0.01
0.14 Ϯ 0.01
0.28 Ϯ 0.01
1.01 Ϯ 0.05
HEPES, pH 7.0 (25 °C)
397 Ϯ 72
431 Ϯ 40
545 Ϯ 38
766 Ϯ 77
(
Table 1 and Refs. 3, 7, 9, 11, and 43). To get a deeper insight in
HEPES, pH 7.0 ϩ 10 mM phosphate (25 °C)
Phosphate, pH 7.0 (25 °C)
Phosphate, pH 7.0 (37 °C)
the inhibition mechanism, the activity of TryS was followed at
variable GSH and fixed saturating co-substrates concentrations
(Fig. 2). The data obtained were fitted to the Haldane’s high
K and K values for GSH (Table 1). Oza et al. (7) reported a K substrate inhibition equation (42) as well as to a more complex
m
i
i
value for GSH that was lower than the respective K value. This equation that takes into account a random-order binding of
m
was not the case in our assays in accordance with a later study substrates to both the catalytically active and inactive enzyme
by Torrie et al. (43). Changing the temperature from 25 °C to and the putative generation of product from the inhibited ternary
3
7 °C resulted in slightly higher K values for all substrates and complex: v ϭ (V
ϩ V
S/K )/(1 ϩ K /S ϩ S/K ϩ1/K K )
m
max1
max2
i
m
i
i 1
shifted the K value for GSH from 143 to 332 ␮M. As expected, (45). In all cases fitting of the kinetic data to the latter equation
i
the strongest effect was on k , which increased nearly 3-fold yielded zero and extremely high values, respectively, for V
cat
max2
Ϫ1
from 1.8 to 5.2 s
.
(activity of the enzyme with GSH bound at the catalytic and
For model construction, TryS was then thoroughly analyzed inhibitory sites) and K (data not shown). This reduced the
1
in the in vivo-like phosphate buffer at pH 7.0 and 37 °C. A pro- equation exactly to the simpler one and strongly suggests that
nounced difference in comparison to HEPES at pH 8.0 was the TryS with GSH bound at an inhibitory site is trapped in an
3
-fold higher K value for GSH. Other major alterations were a unproductive complex. When Gsp served as co-substrate, the
i
rise of the K value for Spd from 139 to 687 ␮M and the reduc- calculated apparent K value for GSH was about 1 mM, compa-
m
i
Ϫ1
tion of the k from 5.2 to 2.8 s . To get a deeper insight in the rable with that obtained in the presence of Spd (Table 1).
cat
underlying mechanism, the kinetic parameters for Spd were
Product Inhibition of TryS by T(SH) —At fixed concentra-
2
assessed in HEPES buffer at pH 8.0 and 7.0 in the presence and tions of ATP and GSH, the presence of T(SH) lowered the
2
absence of 10 mM phosphate and compared with those apparent k and slightly affected the apparent K value for Spd
cat
m
obtained in the in vivo-like buffer (Table 2). This analysis (Fig. 3, A and B). Thus the data were fitted to both non-com-
revealed the pH shift as the main factor for the 5-fold difference petitive and uncompetitive inhibition equations. The separate
in the K value for Spd. The pK values of the amino groups of analysis of each individual experiment did not allow a differen-
m
a
2
3756 JOURNAL OF BIOLOGICAL CHEMISTRY
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Catalytic Mechanism of Trypanothione Synthetase
tiation between both inhibitor types, and a further analysis by
mathematical modeling was required (see below). When GSH
was varied at fixed concentrations of the other substrates,
T(SH) significantly affected the apparent k . Fitting the
2
cat
data to an uncompetitive inhibitor equation yielded a K value
i
for T(SH) of about 360 ␮M, independently if Spd or Gsp was
2
the third substrate (Table 1). Interestingly, at high T(SH) con-
2
centrations the velocity curves for varying GSH adopted a
nearly normal hyperbolic shape (Fig. 3, C and D). With Spd as
third substrate, the calculated K value for GSH was raised from
i
0
.9 mM in the absence to 1.7 and 4.5 mM in the presence of 100
and 560 ␮M T(SH) , respectively. With Gsp as substrate, the K
2
i
value for GSH was raised from 1.1 mM to 2.2 and 4.9 mM when
the assay contained 160 and 560 ␮M T(SH) , respectively. Thus,
2
T(SH) seems to affect GSH binding at its inhibitory site.
2
Time Course of Gsp and T(SH) Production—The activity of
2
TryS in the presence of different concentrations of GSH and
Spd was followed spectrophotometrically. At different times,
an aliquot was removed, and Gsp and T(SH) were quantified
2
by HPLC analysis (Fig. 4). The theoretical NADH consump-
tion was calculated from the sum of Gsp and T(SH) formed.
2
Plotting these theoretical values against the measured
NADH consumption yielded slopes between 0.8 and 1.15
and, thus, a good correlation between ATP turnover and Gsp
and T(SH) production.
2
Independently of the substrate combinations, the synthesis
of T(SH) started with a delay when compared with Gsp forma-
2
tion. During the first minute, T(SH) was practically undetect-
2
able but increased constantly as soon as Gsp had reached a
steady-state level of 60 ␮M (in the presence of 8 mM Spd) or 15
␮
M (at 1.6 mM Spd) (Fig. 4, A and B). The dependence of the Gsp
steady-state level on the Spd concentration is most likely due
to the competition between both polyamine substrates for
enzyme binding. Production of both Gsp and T(SH) was
2
delayed when the reaction mixture contained 2 mM GSH
instead of 0.3 mM GSH (Fig. 4C), in agreement with the very
similar apparent K value for GSH obtained in the presence of
i
either substrate (Table 1). With Spd as substrate, NADH con-
sumption mirrors the total ATP turnover for the production of
Gsp and T(SH) in the two consecutive steps. The ATP con-
2
sumption flux reaches steady state within 1 min after starting
the assay, but the HPLC analysis showed that this reflects a
changing distribution of Gsp and T(SH) produced.
2
TryS Has Negligible ATPase Activity—At concentrations
between 0.8 and 4 mM ATP, a minute and constant ATPase FIGURE 4. Time-dependent production of Gsp and T(SH) . The reaction
2
Ϫ1
mixtures contained 2.1 mM ATP and 170 nM TryS as well as 0.33 mM GSH and
.0 mM Spd (A), 0.33 mM GSH and 1.6 mM Spd (B), and 2.0 mM GSH and 8.0 mM
Spd (C). After different time points, the Gsp (filled circles) and T(SH) (open
activity of Ͻ 0.01 s was observed that corresponded to less
8
than 1% of the synthetase activity. The addition of either 3 mM
2
GSH or 8 mM Spd did not significantly affect the reaction rate. squares) formed were quantified by HPLC analysis as outlined under “Experi-
mentalProcedures.”TheaveragewithS.D. ofthreeindependentexperiments
In the presence of 0.6 mM Gsp, the consumption of ATP
is shown. The insets provide an enlargement of the first 2 min.
increased 25-fold. This does, however, not reflect an increased
ATPase activity but is caused by the amidase activity of the
enzyme (see the next section). The high enzyme concentration
The Amidase Activity of TryS under in Vivo Conditions Is Low—
required for detecting any ATPase activity results in the hydrol- The ability of TryS to catalyze the hydrolysis of Gsp was studied
ysis of Gsp. In the presence of ATP, the GSH and Spd generated at Gsp concentrations between 100 ␮M and 8 mM (Fig. 5). At
by this reaction fuel the synthetase reaction. Indeed, preincu- different time points, the reaction was stopped and GSH and
bation of TryS with Gsp resulted in a rapid increase in ATP Gsp was quantified by HPLC analysis. GSH formation corre-
consumption. Thus, the genuine ATPase activity of TryS is neg- lated with the consumption of Gsp, the sum of GSH and resid-
ligible and independent of the co-substrates.
ual Gsp corresponding to 90–110% of the initial Gsp. Irrespec-
AUGUST 16, 2013•VOLUME 288•NUMBER 33
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Catalytic Mechanism of Trypanothione Synthetase
2
Ϫ1 Ϫ1
tive of the starting Gsp concentration, GSH production was of 9.1 ϫ 10
M
s
that is nearly 2 orders of magnitude lower
4
Ϫ1 Ϫ1
linear during the first 10 min, allowing calculation of initial than that of synthetase reaction (6.6 ϫ 10
M
s
).
velocities from the 5- and 10-min time points. The amidase
For E. coli GspS (46), it has been reported that the synthetase
activity was fitted to a Michaelis-Menten-type kinetic (Fig. 5), substrates, and especially Mg-ATP, highly activate the amidase
Ϫ1
which yielded a k and K values of 5.1 Ϯ 0.7 s and 5.6 Ϯ 1.6 activity toward an artificial substrate. The amidase activity of
cat
m
mM, respectively. Although the k of the amidase activity was T. brucei TryS was studied at a fixed concentration of 0.5 mM
cat
twice that of the synthetase activity (see Table 1), the K value Gsp. The presence of 0.4 or 2.1 mM ATP stopped GSH accumu-
m
lation and triggered the synthesis of T(SH) (data not shown).
for Gsp was almost 200 times higher (5.6 mM compared with
2
Thus, ATP clearly did not stimulate the amidase activity. As the
maximum Gsp concentration used in our kinetic analysis of
TryS was 0.6 mM and all assays contained ATP, the amidase
activity has not been taken into account in the kinetic model.
The Kinetic Model—A kinetic model consisting of linear
mass-action equations for all binding and reaction steps was
constructed for the synthetase activity. To compare putative
mechanisms, we did parameter estimations for different model
versions describing alternative mechanisms. The final model,
which was in best agreement with the data, had an objective
0
.03 mM). Thus, the amidase activity shows a catalytic efficiency
2
value of 3.40 (␮mol/s) and is shown in Fig. 6.
Binding of Substrates to the Enzyme in the Model—In the final
model all three substrates (ATP, GSH, and either Spd or Gsp)
bind to T. brucei TryS before phosphorylation of GSH by ATP
occurs. This is in contrast to the mechanism proposed for the
Crithidia enzyme (3) but in agreement with our steady-state
kinetic analysis that discarded a ping-pong mechanism. Also, in
a ping-pong mechanism, binding of Spd or Gsp after the phos-
FIGURE 5. Hydrolysis of Gsp by TryS. The Gsp hydrolysis in the in vivo-like
buffer system was followed by HPLC analysis. The reaction mixtures con-
tained 1.2 ␮M TryS and 0.1, 0.5, 2, and 7 mM Gsp. The amidase activity as
function of the Gsp concentration was fitted to a hyperbolic kinetic. The aver- phorylation step, would render the K values for ATP and GSH
m
age with S.D. of at least three independent experiments is shown.
independent of whether Spd or Gsp is used as the third sub-
FIGURE 6. Model of the ter-reactant mechanism of TryS catalysis. A, all reactions in the model consist of reversible mass action kinetics, except for those that
generate ADP (reactions 13 and 27). The model includes a “coupling reaction” (reaction 32) that consumes the ADP formed in one (on Gsp) or two (on Spd)
reactions. Compounds connected to the enzyme complex with an underscore (_) represent a complex that cannot proceed in the catalytic cycle, e.g. Gsp that
is formed by the enzyme as product (E_Q) is in a wrong orientation in the substrate cavity for the second reaction. The activated enzyme complexes (XC or XQ)
are the species formed after ATP and GSH reacted to generate a phosphorylated GSH intermediate and ADP. B, the numbers correspond to the steady-state flux
values(␮M/s)obtainedduringasimulationusing1␮M TrySandphysiologicalsubstrateconcentrations(2.3mM ATP, 0.3mM GSH, and8mM Spd). Positivevalues
represent fluxes in the direction of product formation.
2
3758 JOURNAL OF BIOLOGICAL CHEMISTRY
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Catalytic Mechanism of Trypanothione Synthetase
strate. However, our data yielded a K value for GSH that was TABLE 3
m
2
-fold higher on Spd than on Gsp (Table 1). Similar differences Rate constants of the parameterized final model
The values were obtained through parameter estimation as described under “Exper-
were observed for C. fasciculata and T. cruzi TrySs (3, 9). Thus,
imental Procedures.” Constants with the suffix “a” describe the reverse reaction (the
in our model, Spd or Gsp needs to bind before GSH becomes forward direction is in the direction of product formation). For example, the rate of
reaction 1 (Fig. 6A) equals k1 ϫ [E] ϫ [A] Ϫ k1a ϫ [EA]. Reaction 18 corresponded
phosphorylated. Considering the lack of experimental evidence
to the formation of an inhibitory enzyme/T(SH) complex in a previous model. As
2
for a specifically ordered substrate binding, we did not impose
inclusion of this reaction did not improve the model (see “Results”), it was removed.
any predefined order.
Parameter
Value
Unit
Parameter
Value
Unit
␮M sϪ1
Ϫ1
k
2199
s
Ϫ1
TryS produces Gsp as an intermediate in the reactions from
Spd to T(SH) and catalyzes the reaction from Gsp to T(SH)
k
13.5
1
1a
2
16a
17
Ϫ1
␮MϪ1sϪ1
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
134.29
6317
s
k
48.53
2
2
␮MϪ1sϪ1
Ϫ1
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
17a
19
82.5
s
Ϫ1
Ϫ2
Ϫ6
␮MϪ1sϪ1
with Gsp as the direct substrate (Scheme 1). In the HPLC exper-
iments using Spd as substrate (Fig. 4), Gsp accumulated far
above the enzyme concentration of 0.17 ␮M and must, there-
fore, be released from the enzyme. Hence the selected topology
of our kinetic model does not allow the direct production of
2a
3
3a
4
4a
5
5a
6
6a
7
7a
8
8a
9
9a
10
10a
11
11a
12
12a
13
1
14a
15
15a
16
42.45
223.4
286.7
762.3
39.72
22.92
1465
s
1.740 ϫ 10
868.3
␮MϪ1sϪ1
s
Ϫ1
19a
20
Ϫ1
␮MϪ1sϪ1
s
2.005 ϫ 10
␮MϪ1sϪ1
1 ϫ 10
Ϫ10
s
Ϫ1
20a
21
Ϫ1
␮MϪ1sϪ1
s
94.60
␮MϪ1sϪ1
4
Ϫ1
21a
22
5.663 ϫ 10
897.7
s
Ϫ1
␮MϪ1sϪ1
s
Ϫ9
␮MϪ1sϪ1
Ϫ1
6.497 ϫ 10
1 ϫ 10Ϫ10
22a
23
26.8
s
T(SH) from Spd; both Spd and Gsp can compete for binding to
s
Ϫ1
1 ϫ 10Ϫ10
␮MϪ1
s
Ϫ1
2
␮MϪ1sϪ1
1 ϫ 10
Ϫ10
s
Ϫ1
7067
530.5
125.3
237.5
369.7
29.88
261.2
7289
6210
315.7
30.0
23a
24
the enzyme complexes. Structural information (47, 48) from
L. major TryS indicates that the Gsp produced from Spd has to
change orientation in the active site for further catalysis.
Accordingly, our kinetic model clearly differentiates between
Gsp bound as product of the first reaction (E_Q) and as sub-
strate of the second one (EQ; Fig. 6A).
Ϫ1
4
4
␮MϪ1sϪ1
s
5.468 ϫ 10
1.368 ϫ 10
8.612 ϫ 10
␮MϪ1sϪ1
s
Ϫ1
24a
25
Ϫ1
Ϫ14a
␮MϪ1sϪ1
s
␮MϪ1sϪ1
Ϫ10
Ϫ1
25a
26
1 ϫ 10
s
Ϫ1
Ϫ2
␮MϪ1sϪ1
s
4.750 ϫ 10
1058
␮MϪ1sϪ1
s
Ϫ1
26a
27
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1
s
15.18
s
␮MϪ1sϪ1
455.5
s
28
Ϫ1
s
28a
29
290.4
s
Incorporating Inhibition by GSH and T(SH) in the Model—
␮MϪ1sϪ1
4
2
3.025 ϫ 10
200.1
s
Ϫ1
Ϫ1 Ϫ1
1331
2.279
543.5
s
29
30
a
␮M
␮M
s
TryS is inhibited by the substrate GSH and the product T(SH) .
2
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1 Ϫ1
s
234.2
s
Ϫ1
We first scrutinized the experimental data to find out how GSH
inhibition is affected by the presence of other substrates. For
this purpose the activity profiles with GSH as varied substrate
s
1219
s
4
30a
31
Ϫ10
Ϫ10
␮MϪ1sϪ1
1 ϫ 10
s
1 ϫ 10
Ϫ1
34.93
4942
488.9
s
31a
32
420.6
s
Ϫ1 Ϫ1
4
Ϫ1
␮M
␮M
s
1.425 ϫ 10
s
Ϫ1 Ϫ1
s
(
Fig. 2) were plotted for different concentrations of ATP, Spd,
a
Ϫ10
Value lower than 1 ϫ 10
but possible, as this constant was calculated from an
and Gsp. The percentage of inhibition at 2 mM GSH relative to
the maximum activity in the curve was calculated. Inhibition
by GSH was either independent of the other substrates or
even enhanced at increasing substrate concentration (data
assignment rule (see “Experimental Procedures”).
binding of GSH and T(SH) to the free enzyme in addition to
2
binding to the activated enzyme. The parameter estimation led
not shown). Furthermore, the product T(SH) inhibited the
2
2
to a marginally lower objective value of 3.12 (␮mol/s) com-
enzyme without significantly affecting the apparent K val-
m
2
pared with 3.40 (␮mol/s) for the final model (Fig. 6A). How-
ues for Spd or Gsp (Fig. 3, A and B) which excluded competi-
ever, as the dissociation constants (reverse constant/forward
tion between T(SH) and Spd or Gsp. Instead T(SH) appears to
2
2
constant) for GSH and T(SH) at the free enzyme were more
interfere with the binding of GSH at the inhibitory site (Fig. 3, C
2
than two orders of magnitude higher than at the activated
and D). This prompted us to place the inhibitory GSH and
T(SH) on the same enzyme-substrate complex. Because the enzyme complex (not shown), we concluded that inhibitory
2
GSH inhibition was not competitive versus ATP, Gsp, and Spd binding of GSH and T(SH) to the free enzyme was of minor
2
and seemed to be even stimulated by them, we included the physiological importance, and thus, this option was excluded
inhibitory binding after all substrates had been bound to the from the final model.
activated enzyme (XC or XQ; Fig. 6A), resembling an uncom-
Simulations with the Parameterized Model—The final
petitive mechanism. This yielded a very good fit throughout the parameterized model (see Table 3 for the values of all con-
2
data with this model (objective value, 3.40 (␮mol/s) ). Alterna- stants) was used to simulate the experiments to which they
tive options, allowing either only the inhibitory GSH or both were fitted. Key kinetics of the TryS model are shown in Fig. 7.
GSH and T(SH) to bind to the quaternary complexes The complete comparison of model results with all experimen-
2
(
(
EABC/EABQ) yielded objective values of 10.7 and Ͼ100 tal data is available through the SysMO SEEK database and JWS
2
␮mol/s) , respectively. Further proof that these positions online as well as from the authors upon request. The model
are not in agreement with the data came from a scheme that simulations accurately describe the enzyme activity at variable
combined both options, i.e. binding of the inhibitors before substrate concentrations (Fig. 7, A–D). Both in the model and in
and after phosphorylation (to EABC/EABQ as well as the experimental data the maximum activity was slightly lower
XC/XQ). When fitting the data to this model, the forward on Gsp than on Spd. The inhibition of TryS by T(SH) and GSH
2
constants for binding GSH or T(SH) to the quaternary com- was quantitatively reproduced by the model (Fig. 7, E–H) as
2
plexes became extremely low in the parameter estimation. well as the shift of the GSH kinetic profile from a substrate
Thus, these options were removed from the final model.
inhibition curve to a more hyperbolic one in the presence of
Because the steady-state data did not allow a distinction high T(SH) concentrations (Fig. 8).
2
between uncompetitive or non-competitive inhibition mecha-
For an independent validation, the model was compared with
nisms, we also made a model that included specific inhibitory the time course data of Gsp and T(SH) (Fig. 4) that had not
2
AUGUST 16, 2013•VOLUME 288•NUMBER 33
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Catalytic Mechanism of Trypanothione Synthetase
FIGURE 8. Simulation of the T(SH) inhibition of TryS. The activities of TryS
2
measured at variable GSH and T(SH) concentrations at fixed concentrations
2
of 2.3 mM ATP and 8 mM Spd (A) or 0.61 mM Gsp (B) (symbols) (see Fig. 3, C and
D) are depicted together with the model simulation (lines). The reactions con-
tained 0 mM (E, solid line), 0.1 mM (Ⅺ, dotted line), and 0.56 mM T(SH) (ƒ,
2
dashed line) (A) and 0 mM (E, solid line), 0.16 mM (Ⅺ, dotted line), and 0.56 mM
T(SH) (ƒ, dashed line) (B).
2
Steady-state Fluxes in the Model Show That Substrate Bind-
ing Is Partially Ordered—If the binding of the substrates was
completely random, the binding constants of one substrate
would not depend on whether other substrates are already
bound to the enzyme. Although our model topology allows
complete random, strictly ordered, or partially ordered (all con-
stants can have unique values irrespective of the enzyme com-
plex to which a substrate binds), the parameter estimation
resulted in a final model where multiple orders of substrate
binding exist in steady state (Fig. 6B and for more details see
below). We have explicitly tested if a true random mechanism
could accommodate the steady-state data. For this purpose we
made a model with a single kinetic constant for the binding of a
substrate irrespective of the enzyme-complex to which it binds.
Although this model yielded good fits for either the Spd or the
Gsp data alone, we could not find a parameter set that fitted
both the Spd and Gsp data in a single model. The best fit had an
2
overall objective value of 6.15 (␮mol/s) , higher than that of the
2
final model (3.40 (␮mol/s) ).
For analysis of the substrate binding order(s) we compared
the steady-state fluxes along different binding paths, at near-
physiological concentrations of the substrates. Fig. 6B provides
the steady-state fluxes of each binding event in the presence of
FIGURE 7. Comparison of the model simulations with the experimental
data of TryS. Kinetic profiles of various reactions were measured with Spd
(
8
mM Spd, 2.3 mM ATP and 0.3 mM GSH. The fluxes are distrib-
left column) and Gsp (right column) as substrates. The reaction mixtures uted among alternative binding paths, yet certain paths carry a
contained fixed concentrations of 2.3 mM ATP and 0.3 mM GSH (A and B),
.3 mM GSH and 8 mM Spd (C), 0.3 mM GSH and 0.5 mM Gsp (D), 2.3 mM ATP
and 8 mM Spd (E), 2.3 mM ATP and 0.5 mM Gsp (F), 2.3 mM ATP, 0.3 mM GSH,
much smaller flux than others. Interestingly, Spd does not bind
as first substrate but only after at least ATP has been bound.
0
and 8 mM Spd (G), 2.3 mM ATP, 0.3 mM GSH, and 0.5 mM Gsp (H). Dashed Moreover, although the model allows Gsp to bind as first sub-
lines are model simulations, and open circles are experimental data (see
Figs. 1, 2, and 3).
strate, there is a higher flux of binding after GSH. These results
are in line with molecular dynamic simulations by Koch et al.
(
48), which indicate that Gsp and Spd binding is strongly influ-
been used for model fitting. In agreement with the experimen- enced by the occupancy of the ATP or GSH binding sites.
tal data, the model accumulated Gsp far above the enzyme con-
How GSH and T(SH) Inhibit the Enzyme in the Model—In-
2
centration, reaching a steady-state level that was dependent on hibition of the enzyme by GSH and T(SH) can be largely
2
the Spd concentration (Fig. 9, A and B). Moreover, T(SH) for- understood by looking at the dissociation constants K (reverse
2
D
mation exhibited a lag phase and was largely delayed in the constant/forward constant for a reaction; Table 3). Inhibitory
presence of 2 mM GSH (Fig. 9C). Taken together, the simula- binding of GSH and T(SH) results in dead-end complexes that
2
tions showed that the current mathematical model is able to are not part of the catalytic cycle (Fig. 6), and hence these bind-
qualitatively and quantitatively describe the TryS kinetic pro- ing events are in thermodynamic equilibrium when the enzyme
file. However, there are some slight quantitative discrepancies, works at steady state. The K values for GSH as substrate
D
e.g. at the lower GSH concentrations in the presence of 0.56 mM depend on the complex to which it binds (1.3 ␮M for the free
T(SH) (Fig. 8A) and at the later time points in the time simu- enzyme, 0.051 ␮M for EQ, and 599 ␮M for EAC), which is an
2
lations (Fig. 9).
additional demonstration of the non-random binding order.
2
3760 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 33•AUGUST 16, 2013

Catalytic Mechanism of Trypanothione Synthetase
FIGURE 9. Model simulation of the time-dependent production of Gsp and T(SH) . The measured (symbols) and simulated (lines) time-dependent produc-
2
tionofGsp(ƒ, dashedline)andT(SH) (E, dottedline)aredepicted. Thetimeframeshowncorrespondstothatusedinthephotometricassays. Theexperimental
2
assays and simulations contained 0.17 ␮M TryS and 2.1 mM ATP as well as 0.33 mM GSH and 8.0 mM Spd (A), 0.33 mM GSH and 1.6 mM Spd (B), and 2.0 mM GSH
and 8.0 mM Spd (C).
The K values for GSH inhibition of the activated complexes ticular enzyme. We show that the kinetic data of T. brucei TryS
D
(
5.2 ␮M for XC and 4.2 ␮M for XQ) are similar, which suggests a under the in vivo-like conditions described here differ signifi-
binding site that becomes accessible in the activated enzyme cantly from published values (7). The proposed buffer system
irrespective of whether Spd or Gsp is bound.
was designed to reflect the most important physicochemical
The K value for T(SH) binding to XQ is slightly lower (1.7 features of the T. brucei cytosol such as environmental temper-
D
2
␮
M) than the respective value for GSH (4.5 ␮M), reflecting com- ature, pH, ion composition, and total ionic strength. The inten-
petition between GSH and T(SH) for inhibitory binding to the
2
tional simplicity of the system required some compromises.
Although the cytosol contains a high protein concentration, the
proposed buffer does not. The validity of this simplification was
supported by the fact that the activity of TryS was independent
of the presence of macromolecular crowding agents, as was
found previously for yeast glycolytic enzymes (22).
activated enzyme complex. Strikingly, the K value for T(SH)
D
2
binding to XC is very high. Apparently, inhibition at XQ is
sufficient to explain the inhibition by T(SH) and its competi-
2
tion with GSH. From a structural point of view we cannot envi-
sion why T(SH) should only bind to one activated complex and
2
not to the other. Imposing identical constants for binding of
Because the substrates of TryS contain a number of proton-
able groups, the pH value of the buffer was expected to play a
crucial role. The intracellular pH of bloodstream T. brucei is
between 7.0 and 7.2 (33, 34, 49, 50). A three-compartment
model of T. brucei that takes into account separate pH values
for the cytosol, mitochondrion, and endosomal/lysosomal
compartments revealed a cytosolic pH close to 7.0 (33). Indeed
using a buffer at pH 7.0 instead of pH 8.0 affected all kinetic
parameters of TryS. The most significant changes were
observed for the K value for Spd and the K value for GSH,
T(SH) to XC and XQ only slightly increased the objective value
2
2
for this model (3.71 (␮mol/s) ). Thus, we have insufficient data
to quantify the affinity of T(SH) to XC.
2
The very low K value for the complex of T(SH) with the
D
2
free enzyme (7.1 nM) indicates that there is strong binding at the
T(SH) production site. Indeed, at 0.56 mM T(SH) and 0.3 mM
2
2
GSH, the ER complex composed 44% of the total enzyme pool
in steady state, and 19% of enzyme was sequestered as XQ_R.
The contribution of the latter complex is, however, essential, as
m
i
removal of T(SH) binding to both activated enzyme species led
2
which rose by a factor of five and three, respectively. African
trypanosomes multiply in the blood of their mammalian hosts.
In fact, at 37 °C, T. brucei TryS displayed a nearly 3-fold higher
activity compared with 25 °C.
to a model with poor performance in the parameter estimation
2
(
objective value 22.4 (␮mol/s) ).
The distribution over the inhibitory complexes XC_B,
XQ_B, XQ_R, and ER at different GSH concentrations gives
For T. cruzi, T. brucei, and C. fasciculata TrySs, amidase
activities of Ͻ1–5% compared with the respective synthetase
activities have been reported (3, 7, 9). T. brucei TryS hydrolyzed
insight in the competition between GSH and T(SH) inhibition.
2
At 0.56 mM T(SH) and 0.3 mM GSH, as compared with GSH
2
only, the fraction of enzyme in XC_B and XQ_B decreased from
Ϫ1
Gsp with a k of 5.1 s and a K value of 5.6 mM. Thus, at
cat
m
1
0 to 3% in favor of ER (44%) and XQ_R (19%). In the absence of
millimolar Gsp concentrations, the enzyme displays significant
amidase activity, whereas under physiological conditions of
Յ70 ␮M Gsp in T. brucei (for a review, see Ref. 51), the reaction
probably does not play any significant role. The addition of ATP
did not only decrease the amidase activity but even stimulated
T(SH) , increasing the concentration of GSH from 0.3 to 2 mM
2
sequestered the enzyme in XC_B and XQ_B (each ϳ35%). In
the presence of 2 mM GSH and 0.56 mM T(SH) , ϳ50% of the
2
enzyme were sequestered in XC_B and XQ_B together and 27%
of the enzyme in XQ_R (17%) and ER (10%). Although the
enzyme fraction that is sequestered by GSH decreases in the
formation of T(SH) . Thus, our results support the conclusion
2
that the amidase activity in the intact parasite as well as in the
kinetic analysis is negligible. Recently, a TryS double-knock-out
T. brucei cell line that expresses an amidase-dead mutant of the
enzyme was generated. These parasites proliferate in vitro and
are capable to infect mice, although less efficiently than wild-
type cells, which indicates that the amidase function is not
required for viability (13).
presence of T(SH) , the total percentage of inhibitory com-
2
plexes slightly increases when the product is added (from 70 to
7
7%). In conclusion, T(SH) inhibits TryS by remaining bound
2
at its product site and, as it does the inhibitory GSH, by binding
to the activated enzyme complex.
DISCUSSION
Standard kinetic characterizations are usually done under
TryS from different trypanosomatid species has been bio-
conditions optimized to get the maximum activity of the par- chemically characterized (3, 7, 9, 11), but the catalytic mecha-
AUGUST 16, 2013•VOLUME 288•NUMBER 33
JOURNAL OF BIOLOGICAL CHEMISTRY 23761

Catalytic Mechanism of Trypanothione Synthetase
nism is still not completely understood. Therefore, we decided complexes was essential for a good fit of the data, in particular
to measure a full kinetic profile of T. brucei TryS under in vivo- to describe the alleviation of GSH inhibition by T(SH) .
2
like conditions and to integrate the data in a single computa-
The very low K value for binding of T(SH) to the free
D
2
tional model. The data and the model support a rapid equilib- enzyme seems to conflict with its apparent K values. But three
i
rium ter-reactant mechanism where all three substrates need to aspects preclude a direct comparison; (a) T(SH) also inhibits
2
be bound before catalysis starts (Fig. 6). A similar mechanism by binding to the activated enzyme, (b) other substrates present
has been proposed for related ATP-dependent peptide ligases in the reaction compete for the free enzyme and, probably as a
such as C. fasciculata GspS (52, 53) and T. brucei ␥-glutamyl- result of this, (c) in steady state the concentration of free
cysteine synthetase (54). However, for C. fasciculata TryS the enzyme is very low, which tends to diminish the actual ER
initial formation of a quaternary complex between the enzyme concentration.
and the substrates has been ruled out (3). Instead, a ping-pong
In E. coli GspS, GSH cannot only bind at its catalytically com-
mechanism has been suggested where GSH and Mg-ATP react petent site but was also found to partially occupy the Spd site
first to generate a phosphorylated GSH intermediate that sub- (55). There it forms a mixed disulfide with a (non-conserved)
sequently reacts with Gsp and produces T(SH) . This mecha- cysteine residue. This binding site is, however, most probably
2
nism was based on the finding of parallel lines in the reciprocal an accidental trap as it was only observed in the absence of Spd
plots when Gsp was varied in the presence of saturating ATP (55). A molecular dynamic simulation on L. major TryS pre-
and different fixed GSH concentrations that were below both dicts that free GSH could also bind at the Gsp binding pocket,
the K and K values (3). For T. brucei TryS we analyzed a much which would likely impair the binding of Gsp and Spd (48). Our
m
i
broader range of GSH concentrations. When increasing the kinetic data are not in accordance with a competitive inhibition
GSH concentration from 40 ␮M to 2 mM, the lines did not follow versus Spd and Gsp, as increased concentrations of either poly-
the expected order and were only partially parallel. Clearly our amine substrate enhanced the inhibition by GSH instead of
proposed catalytic mechanism does not conflict with an activa- reducing it. Alternative models where inhibitory GSH and
tion of GSH to glutathionyl phosphate as has been proposed for T(SH) bind before ATP hydrolysis could not fit the data.
2
E. coli GspS (55); it only implies that formation of this interme- Hence, our model suggests that both T(SH) and GSH can act
2
diate most likely requires the prior binding of all substrates to as uncompetitive inhibitors which interact with the activated
the enzyme.
enzyme complexes (XC and XQ in Fig. 6). It is thus tempting to
TryS catalyzes the production of both Gsp and T(SH) , and speculate that the GSH/T(SH) inhibitory site becomes only
2
2
different possible mechanisms about how the two reactions accessible after a structural rearrangement of the enzyme due
may be connected have been discussed (3). The crystal struc- to the phosphorylation of GSH during catalysis. Large confor-
ture of L. major TryS together with molecular modeling mational changes upon binding of the substrates have been
approaches shows that the synthetase active site is a triangular proposed for C. fasciculata TryS as well as E. coli GspS (3, 6). To
shaped cavity that accommodates the three substrates roughly summarize, our data suggest TryS is inhibited by T(SH)
2
at the vertices of the triangle (47). There is no evidence for a remaining bound at its product site, and by GSH and T(SH) ,
2
second active site, and Gsp as substrate is postulated to bind to sequestering the activated enzyme in unproductive complexes.
the same site as Spd (47). This is corroborated by recent dock- The precise location of the inhibitory site will require novel
ing and molecular dynamics simulations that identified the structural information.
putative binding site for the glutathione moiety of Gsp (48). As
We emphasize that despite the impressive model perform-
reported previously for the T. cruzi enzyme (10), the reaction of ance, not all parameters are fully constrained by the data. The
T. brucei TryS with GSH and Spd did not immediately yield forward and reverse rate constants of binding processes were
T(SH) . Instead we observed an initial buildup of Gsp that sometimes mutually dependent, implying that we can estimate
2
exceeded the enzyme concentration by a factor of Ͼ100. This is the dissociation constant but not the individual rate constants.
clear experimental evidence that the newly formed Gsp has to The model topology, however, is tightly constrained by the
leave the active site and then binds again as substrate in a dif- data. Various alternative mechanisms could be excluded as the
ferent orientation suited for the second reaction. The fact that corresponding models did not fit the entire dataset. For
our model fits the complete dataset only when we explicitly instance, an early model version with phosphorylation of GSH
represent Gsp generated in the first reaction in an orientation before binding of Spd or Gsp did not accommodate the Gsp and
that does not allow further catalysis (E_Q in Fig. 6) further Spd datasets with a single parameter set. The final model cap-
corroborates this conclusion.
tured the entire steady-state kinetics and was able to predict the
Substrate inhibition of TryS by GSH is a known phenome- product profile of HPLC experiments, which were not used in
non. We found K values for GSH that are well above the K
the model parameterization.
i
m
values. It could, therefore, be envisioned that at low concentra-
We propose that the in vivo-like buffer conditions developed
tions, GSH binds almost exclusively at its substrate binding site, here are adopted by others working on bloodstream form
whereas at higher concentrations it acts also as an inhibitor. As T. brucei. This should facilitate the standardization of data and
shown here, T(SH) is also a powerful inhibitor of T. brucei allow the integration of kinetic data into a single computational
2
TryS and affects the inhibition by GSH. In the model, T(SH)
model describing the parasite metabolism under cellular con-
2
displayed a dual effect. There was significant binding of T(SH)
ditions. The inhibition of TryS by both GSH and T(SH) at
2
2
both to the free enzyme in its production site (ER in Fig. 6) and physiological concentrations suggests that the enzyme is tightly
to the activated enzyme XQ. Including binding to the activated regulated in vivo.
2
3762 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 33•AUGUST 16, 2013

Catalytic Mechanism of Trypanothione Synthetase
1
9. Flohé, L. (2012) The trypanothione system and the opportunities it offers
Acknowledgments—We thank Natalie Dirdjaja for the purification of
recombinant TryS, Dr. Brett Oliver for help with PySCeS, Prof. Pedro
Mendes and Prof. Ursula Kummer and their teams for help with
COPASI, Dr. Shodhan Rao for checking the model script, and all
SilicoTryp partners for stimulating discussions.
to create drugs for the neglected kinetoplast diseases. Biotechnol. Adv. 30,
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94–301
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0. Olin-Sandoval, V., González-Chávez, Z., Berzunza-Cruz, M., Martínez, I.,
Jasso-Chávez, R., Becker, I., Espinoza, B., Moreno-Sánchez, R., and Saave-
dra, E. (2012) Drug target validation of the trypanothione pathway en-
zymes through metabolic modelling. FEBS J. 279, 1811–1833
2
1. Bakker, B. M., Michels, P. A., Opperdoes, F. R., and Westerhoff, H. V.
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