Báo cáo khoa học: Molecular basis of the unusual catalytic preference for GDP/GTP in Entamoeba histolytica 3-phosphoglycerate kinase doc

Molecular basis of the unusual catalytic preference for
GDP/GTP in Entamoeba histolytica 3-phosphoglycerate
kinase
Rusely Encalada
1
, Arturo Rojo-Domı
´
nguez
2
, Jose
´
S. Rodrı
´
guez-Zavala
1
, Juan P. Pardo
3
,He
´
ctor
Quezada
1
, Rafael Moreno-Sa
´
nchez
1
and Emma Saavedra
1
1 Departamento de Bioquı
´
mica, Instituto Nacional de Cardiologı

´
a, Me
´
xico D.F., Me
´
xico
2 Departamento de Ciencias Naturales Unidad Cuajimalpa and Departamento de Quı
´
mica Unidad Iztapalapa, Universidad Auto
´
noma
Metropolitana, Me
´
xico D.F., Me
´
xico
3 Departamento de Bioquı
´
mica, Facultad de Medicina, Universidad Nacional Auto
´
noma de Me
´
xico, Me
´
xico D.F., Me
´
xico
The human parasite Entamoeba histolytica, the causal
agent of amebiasis, relies only on glycolysis for its
ATP supply because it lacks the Krebs cycle and oxi-

dative phosphorylation pathways [1,2]. The glycolytic
enzymes of the parasite are highly divergent from the
enzymes present in the human host; they include an
AMP-inhibited hexokinase [3,4], and the non-allosteric
and pyrophosphate-dependent enzymes phosphofructo-
kinase [4,5] and pyruvate phosphate dikinase [4,6,7],
which replace the functions of the allosteric enzymes
Keywords
ATP ⁄ GTP synthesis; glycolysis; nucleotide
selectivity; parasite; yeast
Correspondence
E. Saavedra, Departamento de Bioquı
´
mica,
Instituto Nacional de Cardiologı
´
a, Juan
Badiano No. 1 Col. Seccio
´
n XVI, CP 14080,
Tlalpan, Me
´
xico D.F., Mexico
Fax: +52 55 5573 0994
Tel: +52 55 5573 2911; ext 1298
E-mail:
(Received 14 November 2008, revised 23
January 2009, accepted 28 January 2009)
doi:10.1111/j.1742-4658.2009.06939.x
Phosphoglycerate kinase (EC 2.7.2.3) catalyzes reversible phosphoryl trans-

fer from 1,3-bisphosphoglycerate to ADP to synthesize 3-phosphoglycerate
and ATP during glycolysis. Phosphoglycerate kinases from several sources
can use GDP ⁄ GTP as alternative substrates to ADP ⁄ ATP; however, the
maximal velocities (V
m
) reached with the guanine nucleotides are  50%
of those displayed with the adenine nucleotides. By contrast,
Entamoeba histolytica phosphoglycerate kinase (EC 2.7.2.10) is the only
reported phosphoglycerate kinase displaying higher activity with
GDP ⁄ GTP and lower affinities for the adenine nucleotides. To elucidate
the molecular basis of the Entamoeba histolytica phosphoglycerate kinase
selectivity for GDP ⁄ GTP, a conformational analysis was carried out on a
homology model based on crystallographic structures of yeast and pig
phosphoglycerate kinases. Some amino acid residues involved in the purine
ring binding site not previously described were detected. Accordingly,
Y239, E309 and V311 were replaced by site-directed mutagenesis in the
Entamoeba histolytica phosphoglycerate kinase gene for the corresponding
amino acid residues present in the adenine nucleotide-dependent phospho-
glycerate kinases and the recombinant proteins were purified. Kinetic anal-
ysis of the enzymes showed that the single mutants Y239F, E309Q, E309M
and V311L increased their catalytic efficiencies (V
m
⁄ K
m
) with ADP⁄ ATP as
a result of both, increased V
m
and decreased K
m
values. Furthermore, a

higher catalytic efficiency in the double mutant Y239F ⁄ E309M was
achieved, which was mainly due to an increased affinity for ADP⁄ ATP
with a concomitant diminished affinity for GDP ⁄ GTP. The main
Entamoeba histolytica phosphoglycerate kinase amino acid residues
involved in the selectivity for guanine nucleotides were thus identified.
Abbreviations
EhPGK, Entamoeba histolytica PGK; PGK, 3-phosphoglycerate kinase; ScPGK, Saccharomyces cerevisiae PGK.
FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS 2037
ATP–PFK-1 and pyruvate kinase in the host [8]. The
importance of glycolysis for parasite survival and the
differences found in the glycolytic enzymes compared
with those of the human host, make this pathway a
suitable target for therapeutic intervention.
In this regard, another remarkable difference in the
amebal glycolytic pathway is found in the first reac-
tion of substrate-level phosphorylation catalyzed by
3-phosphoglycerate kinase (PGK; EC 2.7.2.3). This
43–45 kDa monomeric enzyme, highly conserved dur-
ing evolution, transfers the acyl-phosphate group from
1,3-bisphosphoglycerate to Mg
2+
–ADP to produce 3-
phosphoglycerate and Mg
2+
–ATP, in a fully reversible
reaction under physiological conditions. In the majority
of PGKs characterized to date, from several organisms,
the enzymes show higher or similar affinities for other
purine nucleotides such as GTP or ITP to those
observed with ADP ⁄ ATP; however, the phosphoryla-

tion transfer rates displayed with GTP or ITP are on
average 50% lower than with the adenine nucleotides
[9–13]. In marked contrast, an early study in a partially
purified E. histolytica PGK (EhPGK; EC 2.7.2.10) [14]
demonstrated that this enzyme displayed poor catalysis
with ADP ⁄ ATP as substrates; the cause of this behav-
ior was the higher K
m
values for the adenine nucleo-
tides (at least 10-fold) compared with the K
m
values
exhibited with GDP ⁄ GTP. These differences were
recently confirmed by our research group with the
recombinant purified enzyme, where K
m
values for
ADP and ATP were 12 and 44 times higher than the
K
m
values for GDP and GTP, respectively [4]. To our
knowledge, the higher selectivity towards guanine
nucleotides has only been documented for EhPGK.
In order to advance our understanding of the molec-
ular basis underlying the kinetic differences found in
EhPGK, site-directed mutagenesis analysis was under-
taken on the specific amino acid residues interacting
with the nitrogen base in the nucleotide-binding
pocket. Such residues were identified by conformer
search of their side chains in a predicted 3D model of

the amebal enzyme. Our results indicated that one
single substitution was able to increase catalysis,
whereas two substitutions were necessary to increase
affinity for ADP ⁄ ATP in EhPGK.
Results
Nucleotide specificities of Saccharomyces cerevi-
siae and E. histolytica PGKs in cellular extracts
In order to accurately evaluate differences in the nucle-
otide specificities of the amebal and yeast enzymes in
our kinetic assay conditions, and due to the lack of
commercially available purified yeast PGK, V
m
and
K
m
values were determined under initial velocity condi-
tions in cytosolic cellular extracts of both organisms
(Table 1). For the amebal native PGK, the K
m
values
were similar to those displayed by the wild-type recom-
binant purified enzyme [4] (see Table 2 below) and
confirmed the lower K
m
and higher V
m
values reached
with the guanine nucleotides, as described previously
[4,14]. The K
m

values obtained for the native yeast
enzyme (Table 1) were similar to those reported previ-
ously at pH 6.9–7.0 (ADP, 0.2–0.4 mm; ATP, 0.11–
0.32 mm) and pH 7.5 (ADP, 0.2; ATP, 0.48; GTP,
0.17 mm) [9,10]. Moreover, the yeast PGK in cellular
extracts exhibited 2.5 times higher activity with ATP
compared with GTP, which is in agreement with previ-
ously reported values [9,10]. However, such a differ-
ence in V
m
was not evident when the forward reaction
was determined (Table 1); because of the lack of
reported kinetic data in the forward reaction, a
comparison was not possible. These results established
substantial differences in the purine nucleotide
preferences between the yeast and amebal PGKs.
3D predicted model of EhPGK
Predicted 3D structures of the EhPGK were obtained
by means of modeller software (see Fig. S1 for details
on these models) and molecular operating environ-
ment (moe; ) packages,
using as templates the tertiary structures from yeast [15]
and pig [16,17] PGKs, because of their higher levels of
Table 1. Nucleotide specificities of E. histolytica and S. cerevisiae PGKs in cytosolic-enriched cellular extracts. Values represent the
mean ± SD of titrations made with three independent cellular clarified extracts.
Amebal Yeast
K
m
(mM) V
m

(lmolÆmin
)1
Æmg cellular protein
)1
) K
m
(mM) V
m
(lmolÆmin
)1
Æmg cellular protein
)1
)
GDP 0.07 ± 0.03 41 ± 18 0.37 ± 0.15 38 ± 2
ADP 7.4 ± 3.9 5.5 ± 0.8 0.5 ± 0.17 53 ± 15
GTP 0.016 ± 0.01 4.5 ± 0.6 0.06 ± 0.01 4.0 ± 0.3
ATP 5.0 ± 1.7 1.6 ± 0.06 0.42 ± 0.2 10 ± 1
Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase R. Encalada et al.
2038 FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS
identity (59% and 55%, respectively) and similarity
(73%) with EhPGK. As expected, the resulting models
were highly similar to the templates by using either moe
(Fig. 1A) or modeller (Fig. S1) packages.
Nevertheless, side-chain replacement around the
nucleotide-binding site required a finer modeling
procedure, exploring the conformational space of side-
chain rotamers in the presence of GDP to induce their
fitting. This procedure was programmed in moe in
order to construct and minimize 1000 different combi-
nations of rotamers in the replaced side chains, and

evaluate the resulting diversity. Although the backbone
geometry of the EhPGK models proved to be essen-
tially identical to those of template structures, the vari-
ability in side-chain orientations allowed us to propose
some mutants which might respond to the changes in
the donor ⁄ acceptor of hydrogen bonds in the purinic
ring of adenine respect to guanine nucleotides. It
should be noted that these mutations cannot be
detected by a simple replacement method because
almost none of the 1000 models have all the non-con-
served side chains, which interact with GDP, simulta-
neously oriented in an optimal position. This suggests
that the change in specificity from ATP⁄ ADP to the
guanine nucleotides must be acquired by a cooperative
effect of several amino acid side chains.
Amino acid residues interacting with the guanine
moiety of GDP in EhPGK
The amino acid residues known to interact with the
adenine moiety in the ADP ⁄ ATP-binding site in the
crystal structures of PGKs from several sources have
been identified previously [18] and are illustrated in
Fig. 2. In yeast PGK, these residues correspond to
Gly211, Ala212, Phe289, Leu311, Gly338 and Val339
(Fig. 2A), which lie in a hydrophobic binding pocket
in the C-terminal domain (Fig. 2B).
Based on the two predicted structures of the EhPGK
obtained using the modeller program (Fig. S1),
it was found that the only difference in the amino
acids that bind the purine ring was the presence of Val
instead of Leu at position 311 (Fig. 2A). Based on a

blast analysis, the frequency of Leu at this position is
high, because it was found in almost all PGK amino
acid sequences reported in the Protein Data Bank for
bacteria, fungi, plants and animals (data not shown).
By using a more dynamic method for modeling
EhPGK structure with the moe package in the presence
of GDP, other putative amino acid side chains interact-
ing with the guanine moiety were detected. From this
structural analysis it became evident that the amino
group at carbon 2 of the guanine ring may interact with
the side chain of Glu309, whereas the carbonyl group at
position 6 of the guanine ring may interact with the
hydroxyl group of the Tyr239 side chain (Figs 1B and
2B). In an extended blast analysis to that shown in
Fig. 2A, the more frequent amino acid residue at posi-
tion 309 is Met, although Gln can be found in fungal
PGKs, and Glu or Ser in some bacterial PGKs. By con-
trast, a Phe residue at position 239 was present in almost
all PGK sequences with some exceptions; Tyr was only
found in the PGK structures from Bacillus stearother-
mophilus (1PHP) [19], Trypanosoma brucei (13PK) [20]
Table 2. Kinetic parameters for nucleotides of the wild-type and mutant EhPGKs. Figures indicate mean ± SEM of titrations made with 3–5
independent batches of purified enzymes.
GDP ADP
V
mf
(lmolÆmin
)1
Æmg
protein

)1
) K
m
(lM)
V
mf
⁄ K
m
(LÆmin
)1
Æmg
protein
)1
)
V
mf
(lmolÆmin
)1
Æmg
protein
)1
) K
m
(lM)
V
mf
⁄ K
m
(LÆmin
)1

Æmg
protein
)1
)
Wild-type 820 ± 119 112 ± 20 7.3 280 ± 14 2016 ± 210 0.14
Y239F 1000 ± 161 155 ± 13 6.5 371 ± 55 2693 ± 337 0.14
V311L 969 ± 10 168 ± 23 5.8 562 ± 18
a
1696 ± 168 0.33
E309Q 1250 ± 53
b
158 ± 40 7.9 774 ± 171
b
1723 ± 240 0.45
E309M 1530 ± 269 208 ± 12
b
7.4 1411 ± 350
b
1606 ± 349 0.88
Y239F ⁄ E309M 989 ± 158 802 ± 106
a
1.2 362 ± 64 280 ± 27
a
1.29
GTP ATP
Wild-type 108 ± 12 151 ± 14 0.72 84 ± 9 1439 ± 215 0.06
Y239F 167 ± 29 440 ± 160 0.38 156 ± 12
a
2244 ± 428 0.07
V311L 115 ± 9 189 ± 32 0.61 120 ± 7

b
1104 ± 62 0.11
E309Q 300 ± 13
a
291 ± 66 1.0 315 ± 152 1313 ± 46 0.24
E309M 169 ± 24 153 ± 24 1.1 182 ± 20
a
637 ± 152
b
0.29
Y239F ⁄ E309M 348 ± 69
b
342 ± 22
a
1.0 384 ± 65
a
587 ± 155
b
0.65
One-tailed Student’s t-test for nonpaired samples:
a
P < 0.005;
b
P < 0.05 versus wild-type.
R. Encalada et al. Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase
FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS 2039
and EhPGK. Thus, in order to determine the role of
Y239, E309 and V311 on the GDP ⁄ GTP preference of
EhPGK, the single mutants Y239F, E309M, E309Q and
V311L, and a double mutant Y239F ⁄ E309Q were

generated by site-directed mutagenesis.
Biochemical properties of mutant and wild-type
EhPGK
The mutant proteins were overexpressed as N-terminal
histidine-tailed recombinant proteins in Escherichi-
a coli and purified to a high degree (> 98%; Fig. S2).
No significant differences were found between wild-
type and mutant proteins regarding the following
properties.
Storage stability
The proteins were highly stable when stored in the
presence of 50% glycerol at )20 °C, losing 50% activ-
ity within 4–7 months for the wild-type, Y239F,
Y239F ⁄ E309M and E309Q enzymes. Unexpectedly,
the E309M mutant exhibited a half diminution in
activity only after 12 months (data not shown).
Thermal stability
Wild-type, V311L and the double mutant
Y239F ⁄ E309Q enzymes incubated at 50 °C for
1–12 min did not show drastic reductions in activity,
whereas 30% of their initial activities decayed after
1 min incubation at 60 °C (data not shown). Thus,
inactivation kinetics was carried out at 55 °C (Fig. S3).
The inactivation constants (k
inac
) obtained for wild-
type, V311L and the double mutant were )0.36, )0.41
and )0.35 min
)1
, respectively, which indicated no

significant thermostability differences.
pH activity dependency
The PGK activity dependence determined in the
reverse reaction for the V311L and double-mutant
enzymes showed similar behavior to that of the wild-
type enzyme (Fig. S4).
Oligomeric structure
In a previous study [4], a dimeric oligomeric structure for
the recombinant EhPGK was reported; however, in such
gel-filtration chromatography experiments the protein
fraction recurrently eluted as an entity of intermediate
molecular mass between a monomer and a dimer. By
using a modified protocol, a monomeric, kinetically
active protein was determined for wild-type recombinant
EhPGK; this quaternary structure was not modified in
the V311L and Y239F ⁄ E309M mutant proteins
(Fig. S5). This structural arrangement agrees with the
active monomeric forms of the majority of PGKs from
other sources (either native or recombinant forms)
reported to date. Whether this oligomeric state is
preserved within the amebal cells remains to be explored.
Kinetic parameters in the forward reaction
The V
m
, K
m
and catalytic efficiency (V
m
⁄ K
m

) values
for nucleotides were determined in the forward and
reverse reactions for the wild-type and the five EhPGK
mutants (Table 2). The V
m
in the forward reaction
(V
mf
) and K
m
values with GDP were not greatly
affected in the mutants Y239F, V311L, E309Q and
E309M; thus, no significant change in the catalytic effi-
ciency was observed. However, the double mutant
Y239F ⁄ E309M displayed a sevenfold reduction in its
A
B
Fig. 1. Predicted 3D structure of EhPGK. (A) Overlapped structures
of the pig PGK crystal structure with ATP (1KF0; green) and E. his-
tolytica PGK predicted model with GDP (red) obtained using
MOE software. (B) Close up of the purine ring binding site in the
C-terminal domain. Lines indicate hydrogen bonds interactions. Let-
tering in red corresponds to EhPGK, and green refers to pig PGK.
Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase R. Encalada et al.
2040 FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS
A
B
Fig. 2. Amino acid residues surrounding the purine nucleotide pocket in PGKs. (A) Partial alignment of the C-terminal domain of several
PGKs. The EhPGK amino acid sequence alignment was performed with the
CLUSTALW2 tool and the indicated primary sequences for the other

sources. Numbering on right and left correspond to that used in the text for each species. Marks indicate amino acid residues known to
interact with the nitrogen base groups: circles, conserved residues; down triangles, amino acids changed in EhPGK. Residues in italics indi-
cate the three hydrophobic patches (A, B, C) surrounding the purine base. (B) Schematic diagram showing the amino acid residues located
in the purine base pocket. Interactions between functional groups in the adenine base in pig PGK (upper) and guanine base in modeled
EhPGK (down) were calculated and represented with
MOE. Shadows represent solvent accessibility of ligand atoms and protein residues.
Hydrogen bonds formed between protein and ligand are represented by arrows pointing in the donor to acceptor direction. The hydrogen
bond established by N6 of adenine is either with Gly312 (pig), Leu313 (horse) or Leu311 (yeast) main chain carbonyl group. For yeast PGK
numbering, two amino acid residues should be subtracted in the upper figure.
R. Encalada et al. Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase
FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS 2041
affinity for GDP, which was reflected in a substantial
decrease in the catalytic efficiency with this substrate.
Regarding the values obtained with ADP, the single
mutants V311L, E309Q and E309M exhibited a
tendency to increase both catalytic capacity (V
mf
) and
affinity (decreased K
m
), despite the experimental vari-
ability found by using different protein purifications.
The combined changes in both parameters produced a
concomitant 2.4- to 6.3-fold increment in their cata-
lytic efficiencies with this nucleotide. Remarkably, the
Y239F ⁄ E309M mutant exhibited a significant increase
in its affinity for ADP (7.2-fold), which was not
attained in its respective single mutants, and which
was accompanied by a decrease in its GDP affinity.
Hence, the double mutant yielded a very strong non-

additive increment of its catalytic efficiency of 9.2
times for the phosphorylation of the adenine nucleo-
tide. These observations with the double mutant
indicated the existence of a cooperative effect for GDP
(and ADP) binding, as predicted by molecular model-
ing, because the corresponding single mutants did not
induce significant changes.
Kinetic parameters in the reverse reaction
In general, the single mutants exhibited no significant
differences in the V
m
value in the reverse reaction (V
mr
)
and K
m
values with GTP compared with the wild-type
enzyme, except for a slight increase in the V
mr
of the
E309Q mutant. However, the double mutant displayed
a slight increment in V
mr
and a significant decrease in
the affinity for GTP. The kinetic analysis with ATP
showed that the single mutants Y239F and E309M and
their double mutant Y239F ⁄ E309M significantly
increased (1.9–4.6 times) their V
mr
values. With the

exception of the Y239F mutant, which decreased its
affinity for ATP compared with the wild-type, the other
four mutants displayed a tendency to increase their
affinity for ATP, producing a 1.8–10.8 times increment
in their catalytic efficiencies. Therefore, the increased
V
mr
and decreased K
m
values for ATP were in agree-
ment with the results obtained for ADP in the forward
reaction. It is worth noting that although changes in
catalytic efficiency were not significant for GTP in the
double mutant, a strong cooperative effect on this
parameter was indeed attained for ATP.
The K
m
values for the co-substrate 3-phosphoglycer-
ate were not significantly affected in the mutants
compared with the wild-type enzyme, with K
m
values
(in lm) of 547 (wild-type), 322 (Y239F), 449 (E309Q),
570 (E309M) and 343 (Y239F ⁄ E309M); these values
were obtained by using one protein purification for
each substrate titration.
Nucleotide dissociation constants
Because the K
m
value is by definition a kinetic para-

meter resulting from the V
m
⁄ [V
m
⁄ K
m
] ratio [21], then
the specific nucleotide-binding constant (K
d
) was deter-
mined in the absence of 3-phosphoglycerate for each
nucleotide in the wild-type and the double-mutant
enzymes (because the latter displayed the greatest
changes in nucleotide affinities, as described above).
The true nucleotide substrate of PGKs is the Mg–ATP
complex [10,11]. Therefore, the K
d
values were deter-
mined in the presence of saturating concentrations of
MgCl
2
(Table 3). From these experiments it became
evident that wild-type EhPGK indeed displayed higher
affinity for the couple GDP ⁄ GTP compared to the
couple ADP ⁄ ATP. Unexpectedly, this preference was
also maintained in the Y239F ⁄ E309M mutant.
Discussion
The primary intermediary metabolism of the amitoc-
hondriate parasite E. histolytica differs in several
aspects from that of its human host, as made evident

from the early biochemical studies carried out mainly
by Richard E. Reeves’ laboratory [1,2] and recently by
the genome sequence data analysis [22]. The lack of
the Krebs cycle and oxidative phosphorylation activi-
ties poses glycolysis as the main pathway to generate
ATP for cellular work and as a potential target for
therapeutic intervention [23]. Moreover, the absence of
the typical mammalian flux-controlling glycolytic
enzymes ATP-PFK and pyruvate kinase [24] and the
presence of an AMP-inhibited instead of a glu-
cose 6-phosphate-inhibited hexokinase, emphasizes the
important deviations in the control of the glycolytic
flux in the parasite compared with its host, as recently
demonstrated by metabolic control analysis through
kinetic modeling of the entire pathway [8] and pathway
reconstitution experiments [25].
Table 3. Dissociation constant values for the Mg
2+
-nucleotide
complexes in the wild-type and Y239F ⁄ E309M mutant EhPGKs.
The Mg
2+
concentration was10 mM in the assay. Values represent
the mean of two experiments performed with two purified protein
preparations.
K
d
(lM)
Wild-type Y239F ⁄ E309M
GDP 3 0.51

ADP 11.5 4.5
GTP 0.38 0.32
ATP 10.3 7.7
Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase R. Encalada et al.
2042 FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS
The presence of a guanine nucleotide-dependent
PGK in the first reaction of substrate-level phosphory-
lation of glycolysis most likely changes its interplay
with other metabolic pathways and cellular processes
in the parasite. Saturating concentrations of GDP
(0.7 mm) and GTP (0.8 mm) for EhPGK are found in
E. histolytica trophozoites, whereas non-saturating
concentrations of the adenine nucleotides (3.3 mm
ADP; 5 mm ATP) are present [8], thus suggesting that
the preference for guanine nucleotides of this peculiar
amebal enzyme has physiological relevance. If EhPGK
preferentially synthesizes GTP in vivo, then the pres-
ence of a nucleoside diphosphate kinase, identified at
the genome level [22], should be able to make ATP
readily available from the GTP pool. Moreover,
because E. histolytica lacks a de novo purine synthesis
pathway (the parasite uses instead a purine-salvage
pathway) [26], GTP for DNA synthesis is perhaps
directly supplied by the amebal PGK reaction.
However, these hypotheses have not been yet explored.
All PGK tertiary structures experimentally deter-
mined are highly conserved throughout the taxonomi-
cal groups, mainly because all have a high degree of
similarity (> 50%) at the primary sequence level [27].
As expected, the predicted 3D structures for EhPGK

are very similar to those determined for the yeast and
pig enzymes [15–17], as judged by a RMSD < 1.9 A
˚
for alfa-carbon atoms. The predicted structures can be
considered as high-accuracy models due to the almost
60% identity in their sequences,  95% of their main
chain atoms being expected within 1.5 A
˚
, according to
Baker & Sali [28].
The residues that bind the adenine ring of ADP and
ATP, or those from different adenine nucleotide-based
analogs (AMP–PNP, adenylylimidodiphosphate,
AMP–PCP) or inhibitors (adenylyl 1,1,5,5,-tetrafluor-
opentane-1,5-bisphosphonate), have been identified in
the crystal structures of yeast [15,18], horse [29,30], pig
[16,17,31], B. stearothermophilus [19], T. brucei [20,32]
and Thermotoga maritima [33]. In all these enzymes,
the adenine ring lies in a hydrophobic pocket on the
surface of the C-terminus domain where, as described
for the horse sequence [29], three highly conserved
patches can be identified (Fig. 2): Gly212, Gly213,
Ala214 (patch A); Gly236,Gly237, Gly238 (patch B);
and Val339, Gly340 and Val341 (patch C).
The corresponding reported amino acid residues
involved in adenine binding in the other crystallized
proteins, as well as in the EhPGK identified in the
predicted model, were totally conserved (Figs 1B and
2). In the yeast structure, the adenine base is
positioned on top of Gly338 (patch C), whereas

Gly211 (patch A) and the side chains of Leu311 and
Val339 (patch C) define the limits of the adenine-bind-
ing site [15,18].
It has been reported that N6 of the adenine ring
establishes a hydrogen bond with the main-chain
carbonyl group of Leu311 (yeast) [15], Leu313 (horse)
[29] or Gly312 (pig) [31] amino acid residues (Fig. 2B).
A Gly residue is also present in EhPGK in an equiva-
lent position to that of Gly312 in the pig enzyme. On
the other hand, the Leu residue present in the horse
and yeast enzymes is conserved in the majority of
PGK sequences reported to date; however, in the
EhPGK, it is substituted by a Val residue (Fig. 2A).
The presence of the shorter lateral chain of Val as the
only evident change in the purine-binding pocket site
of the EhPGK suggested that it could accommodate
the larger guanine base; however, the single mutant
V311L displayed no significant changes in its kinetic
parameters with the four purine nucleotides as
compared with the wild-type (Table 2).
Thus, it was necessary to use combinations of side-
chain conformers during modeling to identify other
potential amino acid residues involved in the higher
preference for the guanine nucleotides of the amebal
enzyme. As a result, Tyr239 and Glu309 were identi-
fied in the predicted 3D structure of the EhPGK. They
act as hydrogen bond donor and acceptor, respectively,
to the carbonyl and amino groups present in the guan-
ine ring, at distances of  1.6 A
˚

(Fig. 2B). The
carbonyl, a hydrogen-bond acceptor group of guanine,
is replaced by a donor amino group in adenine,
whereas the guanine amino at position 2 is absent in
adenine (Fig. 2B). Other close contacts are found with
Phe289 and Val311, at distances  2.9 A
˚
from the
guanine ring (Fig. 2B). Among these residues, Phe289
was conserved in all PGK sequences analyzed. The
analysis of the predicted structure suggested that the
presence of the shorter lateral chain of Val311 instead
of Leu allows for the movement of Tyr239 and Glu309
towards the guanine ring, favoring its stabilization by
the two hydrogen bonds just described.
Analysis of the kinetic parameters (K
m
, V
m
, V
m
⁄ K
m
)
with the four purine nucleotides in the single mutants
Y239F, E309M, E309Q and V311L EhPGKs, revealed
a complex interaction between the mutated amino acid
residues and the nucleotides. Although the K
m
values

for ADP ⁄ ATP were slightly lower in the mutants
compared with the wild-type value, the V
m
values
showed a tendency to increase, resulting in an
increased catalytic efficiency of the single mutants with
the adenine nucleotides. The K
m
values for the co-sub-
strate 3-phosphoglycerate were not significantly modi-
fied in any mutant, which was in agreement with the
rapid equilibrium random bi bi kinetic mechanism
R. Encalada et al. Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase
FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS 2043
described for PGK from different sources [10,34,35].
Results with the single mutants suggested that the
mutations might have produced minor rearrangements
in the protein structure (as they were not drastically
affected in its storage and thermal stability or optimal
pH activity); these putative minor structural changes
could have increased the catalysis with the adenine nu-
cleotides at the level of the phosphoryl transfer or the
release of products. In this regard, it has been well
documented that large movements of the two PGK
domains have to occur in order to bring the two sub-
strates together for the phosphoryl transfer reaction
[20,29,32].
In contrast to the single-mutant enzymes, the double
mutant Y239F ⁄ E309M showed both an increased
affinity for the adenine nucleotides and a decreased

affinity for the guanine nucleotide, in addition to a
high increase in the V
m
value with ATP. However, the
K
d
values for ADP ⁄ ATP obtained in the presence of
Mg
2+
were not significantly different from those
observed in the wild-type enzyme. The observed cata-
lytic efficiency changes in the double mutant (Table 2),
accompanied by no significant changes in the equilib-
rium-binding constant (Table 3), might be due to more
prominent conformational constraints in the PGK
reaction [36]. Simultaneous compensating decrements
in entropy and enthalpy of binding may be involved in
yielding a relatively invariable K
d
value. Although this
compensation has been invoked in protein research
[37], it remains controversial [38] and suggests the use
of isothermal titration calorimetry to fully characterize
the thermodynamic parameters of binding [39] as the
subject of a separate investigation.
In this regard, weak interactions in the adenine ring
binding site have been identified in B. stearother-
mophilus [19] and T. brucei PGK structures [32]. For
example, the N6 of the adenine ring establishes a direct
hydrogen bond with the main-chain carbonyl groups of

Ala292 or Ala314, respectively, and another hydrogen
bond mediated by a water molecule is formed with the
hydroxyl group of Tyr223 or Tyr245 in the bacterial
and trypanosomal enzymes. This Tyr residue is equiva-
lent in position to Tyr239 in the amebal enzyme and
which was modified in the double mutant. However, it
has been well documented that there is low activity
with GTP (0.2–0.4% of the activity with ATP) in the
three trypanosomal PGK isoenzymes [40,41]. Also,
B. stearotermophilus PGK can use GTP or ITP as
phosphate donors, although with lesser efficiency than
with ATP (27% and 42% of the activity displayed with
ATP, respectively) [42]. All other PGKs have a Phe
residue in this Tyr position and may or may not show
activity with GTP. Thus, it seems that the presence of a
Tyr residue at this position is not the only prerequisite
for displaying higher catalysis with guanine nucleotides.
These results show the plasticity of a binding site
considered up to now as relatively conserved.
Thus, although the ability to use guanine nucleotides
has been described in the PGK from several sources,
to our knowledge it has only been analyzed in detail in
the present study of the amebal enzyme. Because
changes in affinities and rate velocities of the double
mutant cannot be explained in terms of a simple addi-
tive effect by single mutations, it seems that a coopera-
tive effect of mutations, or interactions, participate in
nucleotide selectivity. The crystal structures of the
wild-type, single- and double-mutant EhPGKs in a
closed conformation will certainly help to clarify the

molecular arrangement in the guanine ring binding site
during the binding of one or the two substrates.
Experimental procedures
Reagents and chemicals
Glyceraldehyde-3-phosphate, ADP, ATP, GDP, GTP,
3-phosphoglycerate, NAD
+
, NADH, EDTA, phenyl-
methanesulfonyl fluoride, trichloroacetic acid, imidazole,
Tris, Mes and Mops were from Sigma (St Louis, MO,
USA); glycerol, potassium phosphate, MgCl
2
and acetic
acid were from JT Baker (Philipsburg, NJ, USA);
dithiothreitol was from Research Organics (Cleveland, OH,
USA); GAPDH was from Roche (Manheim, Germany).
3D EhPGK structural analysis
Only one PGK gene (protein identifier 75.m00170) has been
found in the E. histolytica genome database (http://www.
tigr.org/tdb/e2k1/eha1/). A couple of 3D structures from
the EhPGK amino acid sequence were predicted by using
the program modeller with the PGK structures from
S. cerevisiae (ScPGK; PDB code 3-phosphoglycerateK),
bound to 3-phosphoglycerate and Mg
2+
–ATP [15], and the
pig muscle PGK (PDB code 1HDI), bound to 3-phospho-
glycerate and ADP [16], as templates (see Fig. S1). In
addition, a 3D model was also constructed using moe soft-
ware and the protein moiety of the PGK structure from pig

muscle, bound to 3-phosphoglycerate and Mg
2+
–AMP–
PCP (b,c-methylene-adenosine-5¢triphosphate) as template
(PDB code1KF0) [17].
Amino acid residues known to interact with the purine
ring of ADP ⁄ ATP in PGKs are highly conserved through-
out most of the evolutionary lineages [18]. Protein sequence
alignments were performed by using clustalw2 (http://
www.ebi.ac.uk/Tools/clustalw2/index.html) and blast
( with the EhPGK
Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase R. Encalada et al.
2044 FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS
sequence and PGK sequences from organisms from several
taxa to identify the amino acid frequency in relevant posi-
tions. Most of the conserved positions were also present in
the three predicted 3D models of the EhPGK. By contrast,
non-conserved residues were modeled using the library of
side-chain conformations present in moe. One thousand
different models were constructed, differing only in the
particular combination of side-chain conformers, being each
of them optimized by energy minimization in the presence
of GDP with the CHARMM27 force field [43].
Site-directed mutagenesis, protein
overexpression and purification
Site-directed mutagenesis was performed in the wild-type
gene previously cloned in our laboratory [4] by using the
PCR-based technique of mega-oligonucleotides [44] and the
High Fidelity PCR system (Roche). The 5¢- and 3¢-external
oligonucleotides used contained NdeI and BamHI restriction

sites; internal oligonucleotides were used to introduce the
desired mutations (Table S1). Single mutations were
(EhPGK amino acid sequence numbering) Y239F, V311L,
E309Q, E309M and a double mutant Y239F ⁄ E309M. PCR
products were cloned in the pGEM-T-easy vector (Promega,
Madison, WI, USA) and sequenced to verify the presence of
the mutation and the absence of any other substitution intro-
duced during the PCR. Mutated genes were further cloned in
the pET28 expression vector (Novagen, Madison, WI, USA)
and re-sequenced. The wild-type and mutant proteins were
overexpressed in E. coli BL21DE3pLysS cells (Novagen),
fused to a histidine tag at the N-terminus and then purified
by metal-affinity chromatography as described previously [4].
Purified proteins were concentrated by ultrafiltration to
0.5–2 mg proteinÆmL
)1
and stored at )22 °C in the presence
of 50% glycerol (v ⁄ v). Purity was determined in silver-stained
SDS ⁄ PAGE gels. Protein concentration determination was
made by the standard Lowry method using trichloroacetic
acid-precipitated protein samples to avoid interference by
imidazole from the purification buffer.
E. histolytica and S. cerevisiae cellular extracts
Cytosolic extracts from E. histolytica HM1:IMSS trophozo-
ites were obtained as described previously [8]. S. cerevisiae
strain BY4741 was grown in YPD medium (1% yeast
extract, 2% peptone and 2% glucose) at 30 °C. The cells
were harvested at the end of the exponential growth,
washed once with ice-cold lysis buffer (25 mm Tris ⁄ HCl
pH 7.6, 1 mm EDTA pH 8.0, 5 mm dithiothreitol and

1mm phenylmethanesulfonyl fluoride) and resuspended in
half its wet weight with the same buffer. The cells were
disrupted with glass beads and a clarified extract was
obtained by centrifugation, which was further stored at
)20 °C in the presence of 10% glycerol (v ⁄ v).
Enzymatic assays
The K
m
and V
m
values were determined in coupled assays
with commercial GAPDH (Roche) at 37 °C by monitoring
the absorbance at 340 nm of both the NAD
+
reduction in
the forward (glycolytic) reaction and the NADH oxidation
in the reverse reaction in a spectrophotometer (Agilent,
Santa Clara, CA, USA). The forward enzymatic assay
contained a buffer mixture adjusted at pH 7.0 (50 mm
potassium phosphate and 10 mm each of acetic acid, Mes
and Tris), 5–10 mm MgCl
2,
2mm dithiothreitol, 1 mm
EDTA, 0.5 mm NAD
+
, 1.6–3.2 U of GAPDH, varied
concentrations of GDP or ADP; 3 mm glyceraldehyde-3-
phosphate was added just before the assay and the reaction
was started by adding 0.04–0.2 lg of wild-type or mutant
enzymes. The reverse assay components were similar to

those of the forward assay except that 50 mm imidazole
replaced potassium phosphate and 0.15 mm NADH were
used, with varying concentrations of GTP or ATP, in the
presence of saturating concentrations of the co-substrate
3-phosphoglycerate (6 mm); the reaction was started by
adding 0.4–2 lg of enzyme. For K
m
value determinations,
nucleotide concentrations were varied until the following
highest concentrations were reached, which were saturating
depending on the enzyme: GDP, 2–4 mm; GTP, 3–6 mm;
ADP, 4–11 mm; and ATP, 4–10 mm, and using saturating
concentration of the co-substrate. When determining the
K
m3-phosphoglycerate
of the wild-type and mutant enzymes, the
concentration of GTP present in the assay was at least 10
times their respective K
mGTP
value. PGK K
m
values for
nucleotides in cellular clarified extracts were determined in
the assay described above in the presence of saturating
concentrations of 3-phosphoglycerate (EhPGK 6 mm;
ScPGK, 9 mm) and 0.5–5 lg of cellular protein depending
on the source and the nucleotide. Basal activity in the
absence of specific substrates was always subtracted and the
activity was calculated under initial velocity conditions.
One substrate titration was made for each independent

protein purification ⁄ clarified extract. The concentration of
all the substrates was routinely calibrated. The nonlinear
fitting of the experimental points to the Michaelis–Menten
equation was performed by using origin microcal v 5.0
software.
K
d
values
Dissociation constants were determined at 37 °Cin50mm
Mops pH 7.0, 10 mm MgCl
2
and 0.5 mg of purified protein.
The decrease in protein intrinsic-fluorescence caused by
nucleotide binding was monitored in a spectrofluorometer
(SLM Aminco-Bowman, Rochester, NY, USA) at 290 nm
excitation and 300–400 nm emission. The highest nucleotide
concentrations used in the assay were 2 mm for GDP ⁄ GTP
and 10 mm for ADP ⁄ ATP.
R. Encalada et al. Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase
FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS 2045
Acknowledgements
This work was supported by CONACyT-Me
´
xico
grants 83084 to ES, 80534 to RMS and ‘Acuerdo del
Rector General’ UAM to AR.
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Supporting information
The following supplementary material is available:
Fig. S1. (A) Overlapped structures of the EhPGK
model based on the pig PGK (1HDI) with the corre-
sponding crystal structure. (B) Overlapped structures
of the EhPGK model based on the yeast PGK with
the corresponding crystal structure.
Fig. S2. Silver-stained SDS ⁄ PAGE gel from purified
wild-type and mutant EhPGKs.
Fig. S3. Thermostability of WT and mutant EhPGKs.
Fig. S4. pH dependence on the activity of WT and
mutant EhPGKs on the reverse reaction.
Fig. S5. Gel-filtration analysis.
Table S1. Oligonucleotides used in this study.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the
corresponding author for the article.
R. Encalada et al. Entamoeba GDP ⁄ GTP-dependent phosphoglycerate kinase
FEBS Journal 276 (2009) 2037–2047 ª 2009 The Authors Journal compilation ª 2009 FEBS 2047