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INTRODUCTION |
Entamoeba histolytica along with a number of other
parasitic protists utilizes an unusual form of phosphofructo-1-kinase
(PFK)1 in a central step in
carbohydrate metabolism. This form of PFK employs inorganic
pyrophosphate (PPi) as a phosphoryl donor. Two genes for
PPi-PFK have been described in E. histolytica
(1-3) with a sequence identity between the two proteins of 17%. The sequence of the larger gene, which codes for a protein of ~60 kDa,
has greater identity to the more phylogenetically advanced plant
PPi-PFKs than it does to bacterial PPi-PFKs.
The cDNA of this gene has been expressed in Escherichia
coli and was found to have kinetic properties that were identical
to those of the enzyme isolated from E. histolytica (3). The
pH dependence and apparent substrate affinities of the cloned enzyme
were identical to those of the PPi-PFK in trophozoite
extracts, indicating that the product of the cloned gene accounts for
most if not all of the PFK activity in E. histolytica
trophozoites (3).
The smaller gene, which codes for a 48-kDa protein, has been expressed
in E. coli as a fusion protein that was found to have a much
lower specific activity than that of the larger enzyme (1). Whereas the
60-kDa PFK has been purified from the amoeba, no information concerning
the expression of the 48-kDa protein is available. The 48-kDa PFK
described in the earlier studies is clearly an expressed product in
E. histolytica because it was cloned from a cDNA library
(1). It may have been present in extracts of the organism but did not
copurify with the 60-kDa product or with the activity of
PPi-PFK (3). Furthermore, if a second activity had been
present which represented at least 10% of the total PFK activity, it
would have been detected in native gel electrophoresis.
The problem in attributing a significant role to the 48-kDa protein in
phosphorylation of fructose 6-phosphate (Fru-6-P) is its extremely low
specific activity with PPi as a phosphoryl donor. The
specific activity of the 60-kDa enzyme is about 2,000-3,000 times
higher than that reported for the smaller PFK (3). Thus, if expressed
at the same level in the organism, the smaller PFK would be virtually
undetectable under normal assay conditions for PPi-PFK. One
possibility is that the smaller PFK has a yet to be determined
catalytic activity. Another possibility is that the 48-kDa protein
represents a regulatory protein as one observes in the multisubunit
structure of plant PPi-PFKs (4). In the instance of the
plant enzymes, the catalytic and regulatory subunits copurify. This was
shown to be unlikely regarding the two E. histolytica PFKs
in the earlier study (3) because no 48-kDa protein was present in the
partially purified fractions of the 60-kDa enzyme from E. histolytica.
In the current work, we compare expression of the two forms of E. histolytica PFK in extracts of trophozoites. The 48-kDa PFK has
been purified to homogeneity from both native and recombinant sources
and has been found to have no detectable activity with PPi
as a phosphoryl donor. On the other hand, the enzyme has high activity
with ATP as a phosphoryl donor, but only after prior activation with ATP.
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EXPERIMENTAL PROCEDURES |
Expression Constructs--
Two oligonucleotide primers designed
on the basis of the sequence at the 5'- and 3'-ends of the 48-kDa PFK
gene and containing additional nucleotides at the 5'-ends to generate
NdeI and BamHI restriction sites were used to
amplify by polymerase chain reaction (PCR) a fragment containing the
48-kDa PFK gene from a genomic clone (2). The PCR fragment was then
cloned into the pCR-ScriptTM SK(+) plasmid using the PCR-ScriptTM
cloning kit as directed by the manufacturer (Stratagene, La Jolla,
CA). The plasmid construct was digested with NdeI and
BamHI to isolate the fragment containing the 48-kDa PFK
gene. The digested fragment was then cloned into the complimentary
sites of the pJC45 prokaryotic expression vector (5) (a gift from Dr.
Iris Bruchhaus of the Bernard Nocht Institute for Tropical Medicine,
Hamburg, Germany). The pJC45 expression vector generates a fusion
protein with an additional N-terminal sequence that includes a stretch
of 10 consecutive histidine residues.
To utilize a second expression system, the above PCR-ScriptTM SK(+)
plasmid construct containing the 48-kDa PFK gene was digested with
NdeI and EcoRI and cloned into the complementary
sites of the pALTER-Ex1 plasmid (Promega). The E. histolytica 60-kDa PFK gene cloned into the pALTER-Ex1 has been
described previously (3).
Enzyme Preparation--
The recombinant 60-kDa
PPi-PFK was purified as previously described (1). The
enzyme preparation was homogeneous on the basis of 10% SDS-PAGE. The
enzyme was stored in 50% glycerol at
20 °C. Before being used for
kinetic assays, the enzyme was dialyzed against at least 400 volumes of
150 mM KTes, 1 mM EDTA, pH 7.2.
The pJC45 vector containing the 48-kDa gene was transformed into BL2
(DE3)[pAPlacIQ] E. coli (a gift from Dr. Bruchhaus), and
the bacteria were plated onto LB medium agar plates with 100 µg/ml
ampicillin, 50 µg/ml kanamycin, and 2% (w/v) glucose at 37 °C.
Freshly transformed single colonies were inoculated into LB medium with
100 µg/ml ampicillin, 50 µg/ml kanamycin, and 2% (w/v) glucose and
grown at 37 °C until the bacterial culture reached an
absorbance of 0.2 at 600 nm. After induction for 3 h in the presence of isopropyl-
-D-thiogalactoside, the
recombinant fusion protein was purified using the His-Bind System
(Novagen, Madison, WI) following the manufacturer's recommendations
for native purification of cytoplasmic proteins.
The 48-kDa PFK lacking the N-terminal polyhistidine sequence and
inserted into pALTER-Ex1 was expressed as follows. The plasmid construct was transformed into DF1020 E. coli, which was
grown on LB. After induction by 0.4 mM
isopropyl-
-D-thiogalactoside for 12-24 h at 30 °C,
the cells were harvested by centrifugation at 5,000 × g for 5 min and resuspended in ~2 volumes of ice-cold buffer consisting of 50 mM Tris-HCl, 0.1 mM
EDTA, 14 mM
-mercaptoethanol, pH 7.4 (extraction
buffer). Phenylmethylsulfonyl fluoride was added to the extraction
buffer to a final concentration of 1 mM only during the
extraction step. The cells were lysed by sonication and centrifuged to
remove debris. The supernatant was loaded on a 15-ml column of
N-6-aminohexylcarboxymethyl-ATP-Sepharose (ATP-Sepharose) (6) preequilibrated with the extraction buffer. The column was then
washed with extraction buffer until the absorbance of the flow-through
was below 0.02 at 280 nm. The enzyme was then eluted with extraction
buffer plus 1 mM ATP. Elution fractions were pooled and
concentrated to a volume of ~10 ml, and the enzyme was exchanged
simultaneously into 20 mM Tris-HCl, 0.1 mM
EDTA, 14 mM
-mercaptoethanol, pH 7.2, using a membrane
filtration apparatus. The concentrated protein was then applied to a
Mono Q HR 5/5 anion exchange column on a fast protein liquid
chromatography system (Amersham Pharmacia Biotech) preequilibrated with
the same buffer. The enzyme was eluted with a linear gradient of 0-0.5
M NaCl in the same buffer. The enzyme, which eluted at
~100 mM NaCl, was homogeneous on the basis of 10%
SDS-PAGE. The purified enzyme was stored in 50% glycerol at
20 °C. Prior to kinetic assays, ultrafiltration was used to
exchange the preparation to assay buffer.
The 48-kDa PFK lacking the added N-terminal polyhistidine was also
purified by chromatography using Blue Sepharose (Cibacron Blue F3G-A,
immobilized on Sepharose CL-6B, Sigma). Harvested bacteria were
resuspended in extraction buffer plus 1 mM
phenylmethylsulfonyl fluoride. Cells were then lysed as described above
for ATP affinity column purification. After centrifugation, the
supernatant was loaded onto a 100-ml column of Blue Sepharose
preequilibrated with extraction buffer. The column was then washed with
extraction buffer until the absorbance of the flow-through was below
0.02 at 280 nm. The enzyme was then eluted with extraction buffer
containing 1 mM ATP. Elution fractions were pooled and
concentrated to a volume of ~10 ml, and the buffer was changed
simultaneously to the Mono Q buffer described in the ATP affinity
purification section. The concentrated protein was then purified on a
Mono Q anion exchange column as described for the ATP affinity
purification procedure. The resultant enzyme preparation was
homogeneous on the basis of 10% SDS-PAGE. The enzyme was stored and
prepared for activity analysis as described above. The Blue Sepharose
method yielded ~10 fold greater amounts of pure enzyme per unit
column volume than the ATP-Sepharose procedure. Using Blue Sepharose,
the overall yield from the lysate was ~55%.
Assay of the 60-kDa PFK--
The initial velocity of PFK
activity was determined spectrophotometrically at 30 °C and at pH
7.2 in a medium containing 150 mM KTes, 3 mM
MgCl2, 1 mM EDTA (assay buffer), plus 0.2 mM NADH, and auxiliary enzymes (2-6 units each of
aldolase, triose phosphate isomerase, and glycerol-3-phosphate dehydrogenase).
Assay of the 48-kDa PFK--
To measure activity, the 48-kDa PFK
was first activated by preincubating at standard activation conditions
unless otherwise indicated. The standard activation conditions were 4 µM 48-kDa PFK and 2 mM ATP in 150 mM KTes (pH 7.2), 3 mM
MgCl2, 1 mM EDTA at 30 °C for 30 min. Aliquots of the preincubations were then diluted 10-fold in a
standard dilution buffer (2 mM ATP and 20 mM
Fru-6-P in 150 mM KTes (pH 7.2), 3 mM
MgCl2, 1 mM EDTA) unless otherwise indicated,
and fixed amounts of the dilution were added to assay cuvettes to start
the reaction. The reactions were conducted at standard assay conditions
(1 mM ATP and 20 mM Fru-6-P in the aforementioned assay medium at 30 °C in a 1-ml assay cuvette) unless
otherwise indicated. Activity was determined spectrophotometrically by
measuring the decrease of absorbance at 340 nm. The measured rate of
the first 60 s of the reaction was recorded. For the determination of kinetic constants, one of the two substrates (a nucleoside triphosphate and Fru-6-P) was kept saturated while the other substrate was varied from 0.1 to 10 Km. The magnesium ion
concentration was kept 4 mM higher than the concentration
of nucleoside triphosphates for all assays containing nucleotide to
ensure that virtually all of the nucleotides existed as the magnesium
complex. All nucleoside triphosphate solutions were determined to
contain less than 0.1% PPi. Nucleoside triphosphate
decomposition in the assay cuvette to its nucleoside monophosphate and
pyrophosphate constituents was undetectable.
Kinetic estimates in this study were obtained using unweighted linear
or nonlinear least squares regressions to the Michaelis-Menten and Hill
models using the GraFit graphical analysis program. All assays were
repeated at least twice, and standard errors of intercepts and slopes
were all less than 10%. The kcat values were
calculated assuming one active site existed per subunit and with a
subunit mass of 48 kDa.
ATP-dependent phosphorylation of sugar substrates other
than Fru-6-P was determined by measuring the generation of ADP. The 48-kDa PFK was first activated using standard activation conditions. The enzyme was then diluted 10-fold in 150 mM KTes (pH
7.2), 3 mM MgCl2, 1 mM EDTA, and
identical quantities were added subsequently to assay cuvettes
containing the same components plus 0.2 mM NADH, 1 mM ATP, 5 mM phosphoenolpyruvate (PEP), 5 units
each of lactate dehydrogenase and pyruvate kinase, and 10 mM indicated sugar substrate. The initial velocities were
determined spectrophotometrically by measuring the decrease of
absorbance at 340 nm.
Antibody Preparation and Purification--
Antibodies against
60-kDa PFK and histidine-tagged 47-kDa PFK were raised in New Zealand
White rabbits. Approximately 200 µg of enzyme with adjuvant was
injected at 2, 4, and 8 weeks, and blood was removed 3 days after the
last injection. For further purification where required, each
preparation was purified by passing the polyclonal antibody-containing
serum through a column of the respective PFK linked to CNBr-activated
Sepharose 4B. In the case of the preparation of the 48-kDa PFK
Sepharose column, the enzyme without the histidine tag was used. The
columns were washed extensively with 0.1 M Tris-HCl, 0.3 M NaCl, pH 8.0, until the absorbance of the flow-through
was below 0.01 at 280 nm. Specific antibodies were then eluted
successively in five steps with buffers of decreasing pH from 7.0 to
2.3 containing 150 mM NaCl. Fractions were neutralized
after elution. Specificity of eluted fractions was determined by
Western blot analysis using dilutions of the elution fractions as
primary antibodies. Antibodies against both 48-kDa PFK and 60-kDa PFK
that eluted at pH 5.5 and pH 4.3 had the greatest specificity and were
pooled and used in all subsequent analyses.
Northern Blot and Quantitation of the mRNA Level of the Two
PFK Genes--
E. histolytica total RNA was isolated from
an amoebae cell sediment containing 1-2 × 108 cells
with the Qiagen DNA/RNA isolation kit. Denatured RNA isolated from
trophozoites and RNA markers was then separated on 1.2% agarose gel
and transferred to a nylon membrane. The membrane was then air dried
and exposed to UV light to cross-link the RNA to the membrane. After
prehybridization, membranes were hybridized with 32P-labeled cDNA probes (1 × 106
cpm/ml) prepared by restriction enzyme digestion of the plasmids containing the PFK genes. For quantitation of the mRNA level of the
two PFK genes, slot blot analysis was performed. A standard was
constructed by a series of 2-fold dilutions of the DNA for each of the
PFK genes, beginning from 1 ng to 1/64 ng. The DNA standard and 30-50
µg of E. histolytica total RNA were slot blotted onto
nylon membranes. The membranes were air dried and UV cross-linked. Northern blot was performed as described above. The content of PFK
mRNA within the total RNA was determined by comparing the intensity
of the signal from the total RNA with the DNA standard. The ratio of
the mRNA level of the two PFK genes was determined.
Western Blot Analysis--
Optimal dilution of the
affinity-purified antisera for Western blot was determined by dot blot.
E. coli cell extract and protein molecular weight markers
were used as negative controls. When quantitation was required, a
series of dilutions of known amounts of the two PFKs ranging from 1 to
100 ng was run in adjacent lanes of the gel electrophoresis. Negative
control and protein samples were separated by 10% SDS-PAGE, then
transferred onto nitrocellulose membranes in 25 mM Tris,
200 mM glycine, 20% methanol at 24 V. Washing and
detection were performed by following the instructions of Amersham
Pharmacia Biotech ECL Western blotting protocols using goat anti-rabbit
immunoglobin conjugated with horseradish peroxidase.
E. histolytica Cultivation--
E. histolytica
trophozoites (strain HM-1:IMSS) were grown axenically in TYI-S-33
medium (7) at 35 °C. Routine cultures were maintained in 15-ml
borosilicate glass tubes and transferred every 3 or 4 days. To obtain
sufficient cells for 48-kDa PFK purification, trophozoites were
cultured in 600-ml Nunclon triple flasks (Fisher Scientific).
48-kDa PFK Purification from E. histolytica--
Amoebae from
3-day-old cultures (4 × 108) were detached from the
surface of flasks by chilling at 4 °C for 30 min. Cells were then
harvested by centrifugation at 500 × g for 5 min,
washed twice with phosphate-buffered saline at pH 7.2, and resuspended in 3 ml of the extraction buffer described for ATP affinity
purification of the recombinant enzyme. The cells were then lysed by
sonication. After centrifugation, the lysate was loaded onto 3 ml of an
ATP-Sepharose column preequilibrated with extraction buffer. The column
was washed with 20 volumes of extraction buffer, and the enzyme was subsequently eluted using the same buffer plus 1 mM ATP.
Each elution fraction was analyzed by Western blot for both PFKs.
Molecular Sizing--
Molecular mass determinations were carried
out on a fast performance liquid chromatography system fitted with a
Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech). A standard
curve was constructed by using a mixture containing 200 µg each of
cytochrome C (12.4 kDa), carbonic anhydrase (29 kDa),
glycerol-3-phosphate dehydrogenase (70 kDa), and alcohol dehydrogenase
(150 kDa) in a medium of 20 mM Tris-HCl, 1 mM
EDTA, and 14 mM
-mercaptoethanol, pH 7.2. The standard
mixture, E. coli PFK (142 kDa), and E. histolytica PFK
samples were chromatographed individually using a Superdex 200 column
preequilibrated with the buffered medium plus or minus additions as indicated.
Other Methods--
Gel electrophoresis of proteins was carried
out using a 10% polyacrylamide support according to the system of
Laemmli (8). Protein concentrations were determined by Bradford's dye
binding assay with bovine serum albumin as the standard (9). All
chemicals and enzymes were purchased from Sigma.
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RESULTS |
Purification of the 48-kDa PFK--
In an attempt to repeat the
findings of Bruchhaus et al. (1), who were able to detect
very low PPi-PFK activity with a recombinant 48-kDa PFK
protein bearing a histidine tag, the 48-kDa PFK was prepared as
described in their report. A homogeneous protein with a mass of the
predicted 50 kDa as indicated by SDS-PAGE was purified successfully
(not shown); however no PPi-PFK activity under the
conditions described previously could be detected at any point during
the purification. Because the relatively high concentrations of
imidazole used to elute the enzyme from the nickel column (400 mM) may have denatured the enzyme, the CD spectrum of the
preparation was compared with that of the homogeneous 60-kDa PPi-PFK. The spectra were nearly identical, suggesting that
the global structure of the 48-kDa protein was maintained. All attempts at dialyzing the eluted protein into a lower salt buffer resulted in an
irreversible precipitation. Several other methods of elution from the
nickel column were attempted, including various concentrations of
imidazole and gradients of imidazole and EDTA. However, all of these
methods also failed to produce an enzyme with detectable PPi-PFK activity. The yield of the 48-kDa PFK fusion
protein using this method, however, was sufficient to raise polyclonal
antibodies that were used as a means of detection of the native protein
expressed without the histidine tag during its subsequent purification.
Conventional PFK purification procedures were attempted to
isolate the 48-kDa PFK without the N-terminal histidine tag using the
antibody to follow the 48-kDa protein at each step of the procedure.
The recombinant enzyme did not bind to phosphocellulose, which is
commonly used for the purification of PPi-PFKs (3, 10), under a variety of conditions. No PPi-PFK activity was detected at any point in the purification process or in cell extracts using the assay conditions described by Bruchhaus et al.
(1). The inability to duplicate previously reported activity
measurements and these results suggested that the 48-kDa PFK gene does
not utilize PPi to phosphorylate Fru-6-P and thus prompted
the trial of alternative purification methods.
Because this laboratory commonly uses both
N-6-aminohexylcarboxymethyl-ATP-Sepharose and Blue Sepharose
for the purification of various ATP-dependent PFKs
(11-13), these media were tried with the recombinant 48-kDa PFK. It
was found that the protein bound to both ATP-Sepharose and Blue
Sepharose. The 48-kDa PFK was eluted from both types of medium by
employing 1 mM ATP in the eluting buffer. Subsequent Mono Q
anion exchange chromatography of the eluate from either procedure
yielded homogeneous enzyme with a size by SDS-PAGE equivalent to the
calculated 47.6-kDa mass (not shown). The recombinant enzyme purified
by ATP-Sepharose chromatography was identified by the crude antibodies
that were raised against the purified, histidine-tagged recombinant
48-kDa PFK. For the isolation of specific 48-kDa PFK antibodies,
the ATP-Sepharose-purified enzyme was linked to CNBr-activated
Sepharose as described under "Experimental Procedures."
Catalytic Properties of the 48-kDa PFK Activation--
The
affinity chromatography isolation procedure indicated that the 48-kDa
PFK interacts with ATP. This observation suggested a reexamination of
the activity in the presence of ATP. In such experiments it was
observed that when assays with relatively high concentrations of enzyme
were allowed to proceed for 30 or more min, a very gradual increase in
ATP-dependent activity was observed, suggesting activation
in the assay cuvette. This led to preincubation assays of the enzyme
with various components of the assay mixture. The testing of the assay
components led to the significant finding that
ATP-dependent PFK activity can only be detected when
relatively high concentrations of enzyme and ATP are preincubated
together before adding the enzyme to the assay mixture (details
discussed below). Addition of the same amount of enzyme to the assay
mixture without prior incubation with ATP resulted in no activity even when ATP concentrations in the assay mixture were high. In such cases
no activity is detected because the enzyme concentration in the
reaction mixture is too low to become activated. Consistent with this
hypothesis, when the enzyme is preincubated with ATP at too low an
enzyme concentration, no activity results when adding an equivalent
amount of enzyme as above to the assay mixture.
The dependence of the activation process on the concentrations of
enzyme and ATP is shown in Fig. 1. The
enzyme and ATP concentrations in the preincubation mixtures that result
in half-maximal activity are 0.72 µM and 0.21 mM, respectively. The time course of activation was
measured at saturating concentrations of both ATP and enzyme. Maximal
activity is attained after 5 min of preincubation as shown in Fig.
2A. To determine whether the
temperature of the preincubation had any effect on the resultant rate
of the enzyme, the preincubation mixtures were incubated at various
temperatures before the resultant activity was measured. The
temperature optimum for the preincubation is 30 °C (Fig.
2B). Based on these results, preincubations for all standard
kinetic assays were subsequently conducted using 4 µM
enzyme and 2 mM ATP in 150 mM KTes (pH 7.2), 3 mM MgCl2, 1 mM EDTA at 30 °C and
lasted for at least 30 min.

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Fig. 1.
Enzyme- and ATP-dependent
activation of 48-kDa PFK. After activation, the activity was
measured under standard assay conditions. The data were fitted using
the Michaelis-Menten model to estimate the preincubation enzyme
concentration that achieves half the maximal rate. A, enzyme
concentration dependence of activation. The enzyme was incubated at
concentrations from 0.1 to 28.5 µM in KTes (pH 7.2) assay
buffer containing 10 mM ATP in 20-µl volumes for 30 min
at 30 °C. Fixed amounts of enzyme from the preincubations were then
diluted 10-fold in standard dilution buffer, and identical volumes were
taken from each dilution and added to assay cuvettes. B, ATP
dependence of activation. The enzyme was incubated in KTes (pH 7.2)
assay buffer at fixed concentrations of 4 µM in separate
tubes containing increasing ATP concentrations from 0.1 to 4 mM. Incubations were carried out in 20-µl volumes at
30 °C. Identical amounts of enzyme were taken after 30 min of
incubation from each tube and were assayed under standard assay
conditions. Increasing the incubation time an extra 90 min did not
increase the activation of the enzyme.
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Fig. 2.
Time and temperature dependence of PFK
activation. A, time dependence. The enzyme was first
prepared at 4 µM in assay buffer at 30 °C. ATP was
added to a final concentration of 2 mM, and aliquots of the
activated enzyme were subsequently added to assay cuvettes using the
standard dilution method at time points from 1 s to 2 h after
the addition of ATP. The reactions were then measured under standard
assay conditions. B, temperature dependence. Fixed
concentrations of enzyme at 4 µM were activated at
various temperatures in assay buffer containing 2 mM ATP in
100-µl volumes for 30 min.
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The enzyme concentration dependence of the activation process was
investigated further using polyethylene glycols. PEGs have been shown
to have an associative effect on macromolecular solutes in aqueous
solution without specifically interacting with them (14). Aggregating
systems have been shown to be shifted to higher degrees of association
by increasing PEG concentration (15). Inclusion of PEG in preincubation
mixtures allowed the 48-kDa PFK to be activated at preincubation enzyme
concentrations that were too low to become activated in the absence of
PEG (Fig. 3). The activation process was
enhanced by increasing concentration and size of PEG in the
preincubations, with the enhancement effect peaking at 20% PEG. PEG
apparently encourages native self-association of the 48-kDa PFK into
the activated state by increasing the local protein concentration in
solution.

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Fig. 3.
Effect of PEG on activation of E. histolytica ATP-PFK. Aliquots of 0.05 µM
enzyme were incubated in separate tubes in 150 mM KTes (pH
7.2), 3 mM MgCl2, 1 mM EDTA, with
or without 2 mM ATP for 30 min at 30 °C. Each incubation
contained either PEG 400 or PEG 6000 at concentrations ranging from 0 to 25%. Equal aliquots were then taken from each incubation and added
to assay cuvettes to measure activity.
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The 48-kDa PFK does not require the MgATP complex for activation
because it is activated maximally without Mg2+ in the
preincubation buffer. Maximal activation of the 48-kDa PFK can also
achieved when it is incubated with other nucleotide triphosphates
(Table I). GTP and ITP as well as the
pyrimidines UTP and CTP are all equally as effective as ATP at
activating the enzyme for measuring ATP activity in the resultant assay
mixture. The nonhydrolyzable ATP analog AMP-PNP and ADP also can
activate the enzyme, both being at least 60% as effective as ATP.
Incubation with AMP, the cosubstrate Fru-6-P, and the product
orthophosphate results in no activation at all. Interestingly,
PPi, despite lacking the nucleotide moiety entirely, is
quite capable of activating the 48-kDa PFK to achieve
ATP-dependent activity, being 75% as effective as ATP in a
30-min preincubation.
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Table I
Activation of ATP-PFK by metabolites
Each metabolite (2 mM) was incubated for 30 min at 30 °C
in 150 mM KTes (pH 7.2) and 1 mM EDTA with the
enzyme concentration fixed at 4 µM. From each activator
tube a fixed volume was diluted 10-fold in 150 mM KTes (pH
7.2), 1 mM EDTA, and an identical volume of each dilution
was then added to an assay cuvette within 10 s. The reactions were
measured under standard assay conditions. Values are the mean of at
least three determinations with the standard deviation provided in the
parentheses.
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Inactivation--
Once activated, the enzyme spontaneously
inactivates by simple dilution. This inactivation can be seen during
the PFK assay, where one observes a decrease in the rate about 100 s after the start of the reaction which is the result of the dilution
of activated enzyme from the concentrated preincubation mixture into
the assay. The inactivation proceeds as a first order reaction. To
characterize the dilution effect, the enzyme was activated by
preincubation at the optimal conditions and subsequently diluted in 150 mM KTes (pH 7.2), 3 mM MgCl2, 1 mM EDTA, with or without 2 mM ATP. Aliquots were then taken from each dilution mixture at increasing time points
and added to assay mixtures to measure the activity (Fig. 4). The first order inactivation rate
constant without additions was 0.09 min
1, and
it decreased by nearly half to 0.05 min
1 when
the enzyme was diluted in buffer containing 2 mM ATP and all other preincubation and dilution conditions were identical. Diluting in buffer containing both substrates at concentrations that
produce maximal PFK activity (2 mM ATP and 20 mM Fru-6-P) substantially decreases the rate of
inactivation (not shown). This experiment is complicated by the fact
that the reaction is proceeding under these conditions. The rate of
inactivation measured at early time before significant reaction has
taken place gave a rate constant of 0.016 min
1. As a result of these experiments, all
kinetic assays were performed by diluting the activated enzyme into
assay buffer containing 2 mM ATP and 20 mM
Fru-6-P when dilution was necessary.

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Fig. 4.
Inactivation by dilution. The enzyme was
first activated under standard activation conditions. Aliquots of
enzyme were then each diluted 10-fold in assay buffer (150 mM KTes (pH 7.2), 3 mM MgCl2, 1 mM EDTA) or assay buffer with 2 mM ATP. At time
points from 0 to 120 min, identical amounts of enzyme were assayed
under standard conditions. The rate constants (k) were
calculated as the negative slope of the first order plot of the natural
log of the rate against time. Only time points within the first 20 min
of the ATP-buffer diluted mixture were used because the reaction
reaches an equilibrium after that time. The reversible first order
reaction with ATP was fitted using the first order exponential decay
equation v veq = (Vmax veq)e k't.
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Aggregation State--
The above experiments on activation and
inactivation suggested that the state of polymerization of the molecule
was the determinant of activity. To determine the aggregation state of
the native 48-kDa PFK as well as the activated enzyme, a size exclusion
chromatography experiment was performed. The polymerization state was
determined for the native enzyme, the enzyme activated with ATP, and
the enzyme incubated with the cosubstrate Fru-6-P alone, which does not
activate the enzyme. For the enzyme-ATP experiment, the concentrations of ATP and enzyme which were found to activate the enzyme maximally were used in the preincubation mixtures before chromatography. For the
Fru-6-P experiment, the concentration of Fru-6-P in the preincubation
mixture was 20 mM, which is the concentration determined to
achieve maximal ATP-PFK activity in the kinetic assay (see below). The
results (not shown) of the molecular sizing experiments indicate that
both the unactivated enzyme and the enzyme incubated with Fru-6-P alone
eluted as a single peak at a position similar to that of glycerol-P
dehydrogenase (70 kDa), indicating that the 48-kDa PFK exists as a
dimer under these conditions. The enzyme preincubated with ATP eluted
as a single peak at a position near that of the E. coli
ATP-PFK (140 kDa), suggesting that it exists as a tetramer after activation.
Kinetic Properties--
The 48-kDa PFK was found to be a highly
active ATP-utilizing enzyme with a kcat value of
250 s
1, which is almost three times the
maximum activity of the ATP-dependent activity of E. coli ATP-PFK (16) and about three-fourths the maximum activity of
the PPi-PFK activity of E. histolytica 60-kDa enzyme. No PFK activity (0.01% level of detectability) was observed when PPi (at 2 mM) was used as a phosphoryl
donor in the assay. Also, ATP activity was not inhibited by this
concentration of PPi. To determine if the 48-kDa PFK could
phosphorylate other sugars using ATP as a phosphoryl donor, the
production of ADP was measured when the enzyme was incubated with other
sugar compounds. Fru-1-phosphate, glucose, glucose 1-phosphate, glucose
6-phosphate, mannose, and ribose 5-phosphate could not substitute for
Fru-6-P in the kinase assay.
Similar to many other ATP-PFKs, the 48-kDa PFK shows cooperative
kinetics with respect to Fru-6-P, with a Hill constant
(nH) of 2.3 and a relatively high
Fru-6-P0.5 value of 3.8 mM (Fig. 5A). With regard to ATP, the
kinetic estimates of the E. histolytica 48-kDa PFK compare
favorably with the ATP-PFK from E. coli (16). The 48-kDa PFK
has an apparent affinity for ATP (Km = 0.12 mM) (Fig. 5B) which is higher than that for the
E. coli ATP-PFK (Km = 0.21 mM). The kcat/Km
value with ATP kcat/Km = 2,200) of the 48-kDa PFK is significantly higher than that for the
E. coli enzyme
(kcat/Km = 390) (16). The
presence of PEG in the assay mixture was found to have a modest
effect on the apparent affinity for Fru-6-P. The apparent
Km value for Fru-6-P was ~33% lower with the
inclusion of PEG, with the effect consistent under different
concentrations and sizes of PEG. There was little effect on
cooperativity and on the maximal velocity of the Fru-6-P saturation
profiles (data not shown).

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Fig. 5.
Substrate dependence of E. histolytica ATP-PFK. Assays were performed at pH 7.2 as
described under "Experimental Procedures." A, Fru-6-P
concentration dependence. B, ATP concentration
dependence.
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Although clearly an ATP-utilizing enzyme, the 48-kDa PFK exhibited many
characteristics not typical of ATP-PFKs. The pH optimum was relatively
acidic, which is more characteristic of PPi-PFKs. The
highest activity in the presence of subsaturating Fru-6-P (2.5 mM) was observed between pH 6 and 7 with only 30% activity at 7.5 and 15% activity at 8.5. Activity measurements below pH 6.0 were compromised by the limited activity of one or more of the
auxiliary enzymes used in the coupled assay. In contrast, E. coli and all known mammalian PFKs have alkaline pH optima. Also
unusual was the proficiency of the enzyme in using other nucleotides as
substrates relative to ATP (Table II).
The apparent affinity and the activity at low concentrations of
substrate were even higher for GTP than for ATP. The 48-kDa PFK still
showed cooperativity with Fru-6-P with each of the nucleotides as
cosubstrates, and the apparent affinity for Fru-6-P remained relatively
high with each of the nucleotides tested. In comparison, the E. coli ATP-PFK has kcat/Km
values for GTP and ITP which are an order of magnitude lower than the
value for ATP (16).
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Table II
Kinetic parameters of E. histolytica ATP-PFK
Assays were performed at pH 7.2 as described under "Experimental
Procedures." Apparent Km values for ATP, GTP, and
ITP were determined at 20 mM Fru-6-P. Apparent
Km values for UTP and CTP were determined at 60 mM Fru-6-P. Fru-6-P values were determined at 1 mM ATP, 1 mM GTP, 1 mM ITP, 7 mM UTP, and 7 mM CTP.
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Although the 48-kDa PFK did not require Mg2+ for
activation, it was required for catalytic activity, similar to other
PFKs. Substituting Mn2+ in the assay resulted in only 16%
of the observed activity with Mg2+, whereas no activity was
detectable when substituting with Ca2+ and
Zn2+.
The 48-kDa PFK appears to be inhibited by ATP at high ATP
concentrations. This inhibition was evident at low concentrations of
the cosubstrate Fru-6-P and disappeared at saturating Fru-6-P (Fig.
5B). ATP inhibition has been demonstrated in other ATP-PFKs. Mammalian PFK has a separate ATP inhibitory site (17), whereas E. coli PFK displays mechanism-based, nonallosteric inhibition by ATP
(18). The mechanism of ATP inhibition in the 48-kDa PFK remains to be
elucidated. Cooperativity in the interaction with Fru-6-P increased at
a higher ATP concentration (Fig. 5A). The mechanism of the
cooperative interaction appears to be allosteric, but the mechanism
needs to be resolved. It may be related to association/dissociation behavior, but the failure of PEG to eliminate cooperativity argues against this interpretation. PEP, which is known to inhibit other PFKs
(12, 19), is an inhibitor of the 48-kDa PFK (Fig.
6). PEP decreased the apparent Fru-6-P
affinity substantially, although it had a limited effect on
cooperativity (n) and no effect on ATP binding (Table
III). The steady-state PEP
concentration in E. histolytica is not known.

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Fig. 6.
PEP inhibition of E. histolytica
ATP-PFK. Assays were performed at pH 7.2 in the presence of
1 mM ATP as described under "Experimental
Procedures."
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Table III
Apparent cooperative behavior of E. histolytica ATP-PFK
Assays were performed at pH 7.2 as described under "Experimental
Procedures."
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We investigated many other compounds for their ability to regulate the
activity of the 48-kDa PFK and have tentatively found no other
effectors. Activity with each potential effector was measured both at
half-saturating (2.5 mM) and saturating concentrations (20 mM) of Fru-6-P. The apparent Fru-6-P affinity of the 48-kDa PFK was not affected by the metabolites AMP, ADP, GDP, cAMP,
orthophosphate, sodium ion, ammonium ion, phosphocreatine, citrate,
fructose 2,6-bisphosphate, 3-phosphoglycerate, and glucose 6-phosphate
(all at 1 mM concentrations), metabolites that have been
demonstrated to modulate PFK in other organisms. Other compounds that
were examined for their ability to regulate the 48-kDa PFK included
phosphoglycolate, lactate, calcium ion, and calmodulin. No effects were seen.
mRNA Levels for 60-kDa and 48-kDa PFKs in E. histolytica
Trophozoites--
Total RNA isolated from trophozoites was used for
Northern blots to determine the expression of the two PFKs at the
transcriptional level. Using a cDNA probe of the 48-kDa PFK gene,
blots of the E. histolytica total RNA showed a single band
of about 1.3 kilobases. The blot with the 60-kDa gene probe
showed a single band of ~1.6 kilobases. Both were the expected
size as determined from length of the genes and the short untranslated
region sequences. A slot blot was used to compare the expression of the
two E. histolytica PFK genes at the mRNA level. By
comparing the intensity of the signal from the total RNA with that of
the signal from the standard DNA series as described under
"Experimental Procedures," the amounts of the mRNA of the two
PFK genes within total RNA were compared (not shown). In 30 µg of
E. histolytica total RNA, there were 250 pg of 60-kDa PFK
mRNA and only 16 pg of 48-kDa PFK mRNA. Considering the size of
the two PFK genes, the mRNA level of the 60-kDa PFK gene is about
10 times higher than that of the 48-kDa PFK gene.
PFK Content in E. histolytica Trophozoites--
The relative
quantities of the two PFK enzymes in amoebal extracts were compared by
Western analysis using known amounts of recombinant 48-kDa and 60-kDa
PFKs as standards as described under "Experimental Procedures" (not
shown). In trophozoites the 48-kDa PFK enzyme was present at about
one-tenth the level of the 60-kDa PFK. These data are consonant with
the data on the mRNA levels.
The native 48-kDa PFK enzyme was readily isolated from trophozoite
extracts using chromatography on ATP-Sepharose as described under
"Experimental Procedures." The ATP-Sepharose isolation procedure indicated no apparent association between the two PFKs in trophozoites. The 60-kDa PFK was never detected by Western blot at any point during
chromatography except in the initial effluent containing proteins that
do not bind to ATP-Sepharose. The search for possible interaction
between the two PFKs was motivated by the observation of a multisubunit
structure in plant PPi-PFKs (4). In the instance of the
plant enzymes, catalytic and regulatory subunits copurify. No
copurification was observed, nor was coprecipitation of the two enzymes
from trophozoite extracts seen when either specific antibody was used.
Furthermore, assays of purified 60-kDa PPi-PFK were not
influenced by the presence of an equal amount of purified 48-kDa
ATP-PFK, nor was there any effect when the two enzymes were
preincubated together. Similarly, no effect of 48-kDa PFK was seen when
the reverse experiments were performed. Thus any direct interactions
between the two proteins are very unlikely.
A reinvestigation of trophozoite extracts showed that ATP-PFK activity
could be detected without prior activation. ATP-dependent PFK activity in amoebae is about 11 fold lower than
PPi-dependent activity (0.43 unit of ATP
activity versus 4.1 units of PPi activity in 100 µl of trophozoite extract), corresponding to the relative amounts of
the two PFKs enzymes detected by Western analysis. To ensure that the
measured ATP-PFK activity was not an artifact of the 60-kDa PFK
catalyzing PPi produced in other metabolic pathways, amoebal extracts were dialyzed exhaustively to eliminate all small metabolites. Also, ATP-PFK activity was readily measured at high Fru-6-P concentrations (20 mM) and was totally undetectable
at 1.5 mM Fru-6-P, which is a saturating concentration of
the sugar phosphate for the 60-kDa PFK. This indicated that the ATP-PFK activity detectable only at the higher Fru-6-P concentration was not
measuring the 60-kDa PFK catalyzing contaminating PPi
because such contamination would have been detectable at 1.5 mM Fru-6-P. In the study that first identified the
PPi-PFK enzyme of E. histolytica, Reeves
et al. (20) also detected ATP-PFK activity in trophozoite homogenates. Those investigators were unable to characterize the E. histolytica ATP-PFK activity further because of activity
losses during purification. In fact, Reeves later concluded that the observed ATP-PFK activity was an artifact (21). That is clearly not the
case as demonstrated here.
An interesting finding was that the amoebal ATP-PFK activity was not
increased by preincubation of amoebal extracts with 2 mM
ATP even after eliminating the small metabolites by dialysis. In
contrast, the trace of ATP-PFK activity in bacterial extracts containing recombinantly expressed E. histolytica 48-kDa PFK
was increased dramatically after incubation with 2 mM ATP
(data not shown).
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DISCUSSION |
The two PFKs of E. histolytica display distinct
phosphoryl donor specificities. The 60-kDa PFK is a
PPi-dependent enzyme and is responsible for all
detectable PPi-PFK activity in trophozoite extracts (3).
The 48-kDa PFK, contrary to previous reports, demonstrates no
detectable PPi-PFK activity when produced recombinantly. The 48-kDa PFK is in fact a highly active ATP-utilizing PFK that is
also able to use other nucleotides efficiently for catalysis. However,
the apparent Km value for Fru-6-P of the 48-kDa PFK
is more than 20-fold greater than previously measured intracellular Fru-6-P concentrations (0.16 ± 0.06 mM) in amoebae
(20), indicating that without a positive effector this enzyme may have
limited physiological activity unless one invokes compartmentalization. Considering that the 60-kDa PFK has been shown to account for the
glycolytic flux in amoebal extracts (21) and that mRNA, protein,
and activity levels all indicate that the 60-kDa PFK is present in
trophozoites in 10-fold greater amounts than the 48-kDa enzyme, the
significance of the ATP-PFK in the glycolysis of trophozoites remains
in question. However, E. histolytica has a complex life
cycle, and the ATP-PFK may have functions in other stages of that cycle.
The findings in this study as well as results from earlier studies from
this laboratory (3) argue against the possibility of the two PFKs of
E. histolytica associating or affecting each other in some
regulatory manner. The two PFKs do not copurify when isolating either
protein, a protein 47 kDa in size was not seen in the active PFK
fractions during native PPi-PFK purification, immunoprecipitation of the trophozoite cell extract with antibodies against the 60-kDa PFK did not precipitate a protein close to 47 kDa in
mass, and the activity of either the 60-kDa PFK or 48-kDa PFK was
unaffected by the presence of its PFK counterpart in the assay mixture
or in preincubations (data not shown).
E. histolytica 48-kDa PFK is an unusual ATP-utilizing PFK
that is only active after incubation at high enzyme concentrations with
ATP. This activation appears to be the result of a change in the state
of aggregation of the enzyme upon binding ATP rather than a catalytic
event such as ATP hydrolysis or the formation of a phosphoenzyme
complex. The activation can be reversed by simply diluting the
enzyme-ATP incubation, and the enzyme-ATP preincubation mixture
eventually reaches an equilibrium. These results indicate that
activation does not involve a permanent alteration in either the enzyme
or the ATP molecule. The enzyme can also be activated by other
nucleotide triphosphates, AMP-PNP, ADP, and even PPi,
indicating that a specific ATP modification is not involved in
activation. These observations suggest that the nucleoside moiety is
not essential for activation and that the last two phosphoryl groups of
ATP are the most critical features. Closer analysis reveals
PPi to be a better activator than ADP and AMP-PNP, both of
which deviate from ATP in the terminal polyphosphate region. This
polyphosphate moiety is completely absent in AMP and orthophosphate,
and incubation with these compounds does not activate the enzyme at
all. The Michaelis constant value for ATP derived from the
ATP-dependent activation assay (Km = 0.21 mM) is similar to that observed from the substrate
dependent assay (Km = 0.12 mM), which is
consistent with ATP binding at the same site for both activation and
catalysis. However, our results show that the adenosine moiety seems to
be of little relevance in activation, whereas PPi activates
the enzyme to nearly maximum levels. Although PPi can
activate the enzyme, it is not a substrate, nor can it inhibit ATP
activity. These results introduce the possibility that the 48-kDa PFK
may bind PPi for activation at a site other than the
substrate binding site. It is also possible that the activators may
produce their effects by chelating some unknown inhibitor; however,
this situation is unlikely because all activation assays were performed
in the presence of 1 mM EDTA. Although the enzyme cannot be
activated by the cosubstrate Fru-6-P alone, the sugar phosphate does
provide some protection against inactivation when present with ATP.
The dependence of the activation process on protein concentration
suggests that the reversible activity loss is associated with
association-dissociation behavior of the protein. This was supported by
experiments with molecular crowding with PEG which showed that crowding
increased the rate of activation. Finally, molecular sizing experiments
show that the inactive PFK exists as a dimer that associates into an
active tetramer upon incubation with ATP.
The 48-kDa PFK in dialyzed amoebal extracts was found in an activated
state. This information introduces many new possibilities for the
native activation state of the enzyme. Although it is possible that ATP
was not eliminated from the trophozoite extracts by dialysis, it is
more likely that another activator exists in the amoeba which is either
too large or too tightly associated to be removed by dialysis. Also,
the 48-kDa PFK may be activated in trophozoites by some other means
such as subcellular localization. Whatever the mechanism of native
activation, it is likely that in vivo the 48-kDa PFK
displays substantially different kinetic features.
The two PFKs of E. histolytica have a low sequence identity
of about 17%, although there are many identical residues in the presumed active site. Phylogenetic studies of the sequences of PFKs
place the two E. histolytica PFKs in a large group of
proteins, most of which have been described as PPi-PFKs
that are distinct from the typical ATP-PFKs such as those found in
E. coli as well as all mesozoans. The 60-kDa enzyme falls
into a monophyletic subgroup that contains a number of other well
characterized PPi-PFKs including those of plants (22, 23).
On the other hand, the 48-kDa PFK sequence from E. histolytica falls into a monophyletic subgroup within the
PPi-PFK group that also contains Treponema pallidum and Borrelia burgdorferi and the peroxisomal
ATP-PFK of Trypanosoma brucei (22, 23). Of the other three
members of the group, the T. pallidum gene product has not
been characterized and preliminary studies of the B. burgdorferi product have not found either ATP- or
PPi-PFK activity (24). The T. brucei ATP-PFK is
a homotetramer with a subunit mass of 50 kDa and is not regulated by
the metabolites that modulate the activity of ATP-PFKs in other organisms (25). The members of this group of four proteins have a
common sequence in the presumed region where the phosphoryl transfer
reaction takes place. The two sequences are GGDG and PKTIDND, which may be contrasted to GGDD and
PKTIDND of almost all well characterized
PPi-PFKs and GGDG and PGTIDND in
ATP-PFKs of E. coli and all mesozoans. Recently we have
shown that mutation of the second Asp in the GGDD sequence of the
E. histolytica 60-kDa PPi-PFK to Gly changes the
specificity to that of an ATP-PFK (26). The last residue in the GGDG
sequence would appear to be a particularly important determinant of the
phosphoryl donor specificity of all PFKs.