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
Trang 1GDP/GTP in Entamoeba histolytica 3-phosphoglycerate
kinase
Rusely Encalada1, Arturo Rojo-Domı´nguez2, Jose´ S Rodrı´guez-Zavala1, Juan P Pardo3, He´ctor Quezada1, Rafael Moreno-Sa´nchez1and Emma Saavedra1
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 diphosphofructo-kinase [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: emma_saavedra2002@yahoo.com
(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 (Vm) 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 (Vm⁄ Km) with ADP⁄ ATP as
a result of both, increased Vm and decreased Km 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.
Trang 2ATP–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 phosphorylareac-tion 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 Mg2+–ADP to produce
3-phosphoglycerate and Mg2+–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 Km values for the adenine
nucleo-tides (at least 10-fold) compared with the Km values
exhibited with GDP⁄ GTP These differences were
recently confirmed by our research group with the
recombinant purified enzyme, where Km values for
ADP and ATP were 12 and 44 times higher than the
Kmvalues 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, Vm and
Kmvalues were determined under initial velocity condi-tions in cytosolic cellular extracts of both organisms (Table 1) For the amebal native PGK, the Km values were similar to those displayed by the wild-type recom-binant purified enzyme [4] (see Table 2 below) and confirmed the lower Km and higher Vmvalues reached with the guanine nucleotides, as described previously [4,14] The Km 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 Vm 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; http://www.chemcomp.com) 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.
Km(m M) Vm(lmolÆmin)1Æmg cellular protein)1) Km(m M) Vm(lmolÆmin)1Æmg cellular protein)1)
Trang 3identity (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.
Vmf(lmolÆmin)1Æmg
protein)1) K m (l M)
Vmf⁄ K m (LÆmin)1Æmg protein)1)
Vmf(lmolÆmin)1Æmg protein)1) K m (l M)
Vmf ⁄ K m (LÆmin)1Æmg protein)1)
One-tailed Student’s t-test for nonpaired samples: a P < 0.005; b P < 0.05 versus wild-type.
Trang 4and 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 coliand 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 50C for 1–12 min did not show drastic reductions in activity, whereas 30% of their initial activities decayed after
1 min incubation at 60C (data not shown) Thus, inactivation kinetics was carried out at 55C (Fig S3) The inactivation constants (kinac) 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 Vm, Km and catalytic efficiency (Vm⁄ Km) values for nucleotides were determined in the forward and reverse reactions for the wild-type and the five EhPGK mutants (Table 2) The Vm in the forward reaction (Vmf) and Km 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.
Trang 5B
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 loindi-cated
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.
Trang 6affinity 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 (Vmf) and
affinity (decreased Km), 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 Vmvalue in the reverse reaction (Vmr)
and Kmvalues with GTP compared with the wild-type
enzyme, except for a slight increase in the Vmr of the
E309Q mutant However, the double mutant displayed
a slight increment in Vmrand 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 Vmr 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
Vmr and decreased Km 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 Kmvalues for the co-substrate
3-phosphoglycer-ate were not significantly affected in the mutants
compared with the wild-type enzyme, with Km 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 Km value is by definition a kinetic para-meter resulting from the Vm⁄ [Vm⁄ Km] ratio [21], then the specific nucleotide-binding constant (Kd) 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 Kd values were deter-mined in the presence of saturating concentrations of MgCl2 (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 m M in the assay Values represent the mean of two experiments performed with two purified protein preparations.
Kd(l M)
Trang 7The 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 (Km, Vm, Vm⁄ Km) 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 Km values for ADP⁄ ATP were slightly lower in the mutants compared with the wild-type value, the Vm values showed a tendency to increase, resulting in an increased catalytic efficiency of the single mutants with the adenine nucleotides The Kmvalues 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
Trang 8described 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 Vmvalue with ATP However, the
Kd values for ADP⁄ ATP obtained in the presence of
Mg2+ 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 Kd 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, MgCl2 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 Mg2+–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 Mg2+–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 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the EhPGK
Trang 9sequence 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)1and 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 30C 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
1 mm 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 Km and Vmvalues were determined in coupled assays with commercial GAPDH (Roche) at 37C 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 MgCl2, 2 mm 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 Km 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
Km3-phosphoglycerateof the wild-type and mutant enzymes, the concentration of GTP present in the assay was at least 10 times their respective KmGTP value PGK Km 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
Kdvalues Dissociation constants were determined at 37C in 50 mm Mops pH 7.0, 10 mm MgCl2and 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
Trang 10This work was supported by CONACyT-Me´xico
grants 83084 to ES, 80534 to RMS and ‘Acuerdo del
Rector General’ UAM to AR
References
1 Reeves RE (1984) Metabolism of Entamoeba histolytica
Schaudinn, 1993 Adv Parasitol 23, 105–142
2 McLaughlin J & Aley S (1985) The biochemistry and
functional morphology of Entamoeba J Protozool 32,
221–240
3 Reeves RE, Montalvo F & Sillero A (1967) Glucokinase
from Entamoeba histolytica and related organisms
Biochemistry 6, 1752–1760
4 Saavedra E, Encalada R, Pineda E, Jasso-Cha´vez R &
Moreno-Sa´nchez R (2005) Glycolysis in Entamoeba
histolytica: biochemical characterization of recombinant
glycolytic enzyme and flux control analysis FEBS J
272, 1767–1783
5 Reeves RE, Serrano R & South DJ (1976)
6-Phospho-fructokinase (pyrophosphate) Properties of the enzyme
from Entamoeba histolytica and its reaction mechanism
J Biol Chem 251, 2958–2962
6 Reeves RE (1968) A new enzyme with the glycolytic
function of pyruvate kinase J Biol Chem 243, 3202–3204
7 Saavedra-Lira E, Ramı´rez-Silva L & Pe´rez-Montfort R
(1998) Expression and characterization of recombinant
pyruvate phosphate dikinase from Entamoeba
histolyti-ca Biochim Biophys Acta 1382, 47–54
8 Saavedra E, Marı´n-Herna´ndez A, Encalada R, Olivos
A, Mendoza-Herna´ndez G & Moreno-Sa´nchez R
(2007) Kinetic modeling can describe in vivo
glycolysis in Entamoeba histolytica FEBS J 274,
4922–4940
9 Krietsch WK & Bu¨cher T (1970) 3-Phosphoglycerate
kinase from rabbit skeletal muscle and yeast Eur J
Biochem 17, 568–580
10 Scopes RK (1973) 3-Phosphoglycerate kinase In The
Enzymes, Vol 8 (Boyer PD, ed.), pp 335–351
Academic Press, New York, NY
11 Lee Ch-S & O¢Sullivan W (1975) Properties and
mechanism of human erythrocyte phosphoglycerate
kinase J Biol Chem 250, 1275–1281
12 Kuntz GWK & Krietsch WKG (1982)
Phosphoglycer-ate kinase from animal tissue Meth Enzymol 90,
103–110
13 Kuntz GWK & Krietsch WKG (1982)
Phosphoglycer-ate kinase from spinach, blue–green algae, and yeast
Meth Enzymol 90, 110–114
14 Reeves RE & South DJ (1974) Phosphoglycerate kinase
(GTP) An enzyme from Entamoeba histolytica selective
for guanine nucleotides Biochem Biophys Res Commun
58, 1053–1057
15 Watson HC, Walker NPC, Shaw PJ, Bryant TN, Wendell PL, Fothergill LA, Perkins RE, Conroy SC, Dobson MJ, Tuite MF et al (1982) Sequence and structure of yeast phosphoglycerate kinase EMBO J 1, 1635–1640
16 Szila´gyi AN, Ghosh M, Garman E & Vas M (2001) A 1.8 A˚ resolution structure of pig muscle 3-phosphoglyc-erate kinase with bound MgADP and 3-phosphoglycer-ate in open conformation: new insight into the role of the nucleotide in domain closure J Mol Biol 306, 499– 511
17 Kova´ri Z, Flachner B, Na´ray-Szabo G & Vas M (2002) Crystallographic and thiol-reactivity studies on the com-plex of pig muscle phosphoglycerate kinase with ATP analogues: correlation between nucleotide binding mode and helix flexibility Biochemistry 41, 8796–8806
18 Joao HC & Williams RJP (1993) The anatomy of a kinase and the control of phosphate transfer Eur J Biochem 216, 1–18
19 Davies GJ, Gamblin SJ, Littlechild JA, Dauter Z, Wilson KS & Watson HC (1994) Structure of the ADP complex of the 3-phosphoglycerate kinase from Bacillus stearothermophilusat 1.65 A˚ Acta Crystallogr
50, 202–209
20 Bernstein BE, Michels PAM & Hol WGJ (1997) Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation Nature
385, 275–278
21 Northrop DB (1999) So what exactly is V⁄ K, anyway? Biomed Health Res 27, 250–263
22 Clark CG, Alsmark UCM, Tazreiter M, Saito-Nakano Y, Ali V, Marion S, Weber C, Mukherjee C, Bruchhaus I, Tannich E et al (2007) Structure and content of the Entamoeba histolyticagenome Adv Parasitol 65, 51–190
23 Saavedra-Lira E & Perez-Montfort R (1996) Energy production in Entamoeba histolytica: new
perspectives in rational drug design Arch Med Res
27, 257–264
24 Moreno-Sa´nchez R, Saavedra E, Rodrı´guez-Enrı´quez S
& Olı´n-Sandoval V (2008) Metabolic control analysis: a tool for designing strategies to manipulate metabolic pathways J Biomed Biotechnol, doi: 10.1155/2008/ 597913
25 Moreno-Sa´nchez R, Encalada R, Marı´n-Herna´ndez A
& Saavedra E (2008) Experimental validation of meta-bolic pathway modeling An illustration with glycolytic segments from Entamoeba histolytica FEBS J 275, 3454–3469
26 Albach RA (1989) Nucleic acids of Entamoeba
histolyti-ca J Protozool 36, 197–205
27 Fleming T & Littlechild J (1997) Sequence and structural comparison of thermophilic phosphoglycerate kinases with a mesophilic equivalent Comp Biochem Physiol A 118, 439–451