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Journal of Bacteriology, January 2001, p. 709-715, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.709-715.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Phosphoenolpyruvate Synthetase from the
Hyperthermophilic Archaeon Pyrococcus furiosus
Andrea M.
Hutchins,
James F.
Holden, and
Michael W. W.
Adams*
Department of Biochemistry and Molecular
Biology and Center for Metalloenzyme Studies, University of
Georgia, Athens, Georgia 30602
Received 21 March 2000/Accepted 25 October 2000
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ABSTRACT |
Phosphoenolpyruvate synthetase (PpsA) was purified from the
hyperthermophilic archaeon Pyrococcus furiosus. This enzyme
catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate
(PEP), AMP, and phosphate and is thought to function in
gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and
contains one calcium and one phosphorus atom per subunit. The active
form has a molecular mass of 690 ± 20 kDa and is assumed to be
octomeric, while approximately 30% of the protein is purified as a
large (~1.6 MDa) complex that is not active. The apparent
Km values and catalytic efficiencies for the
substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and
1.43 × 104 mM
1 · s1 and 0.39 mM and 3.40 × 103
mM1 · s1, respectively. Maximal
activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4).
The enzyme also catalyzed the reverse reaction, but the catalytic
efficiency with PEP was very low
[kcat/Km = 32 (mM
· s)1]. In contrast to several other
nucleotide-dependent enzymes from P. furiosus, PpsA has an
absolute specificity for ATP as the phosphate-donating substrate. This
is the first PpsA from a nonmethanogenic archaeon to be biochemically
characterized. Its kinetic properties are consistent with a role in
gluconeogenesis, although its relatively high cellular concentration
(~5% of the cytoplasmic protein) suggests an additional function
possibly related to energy spilling. It is not known whether
interconversion between the smaller, active and larger, inactive forms
of the enzyme has any functional role.
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INTRODUCTION |
A number of unique microorganisms
that thrive at temperatures of 90°C or higher have been isolated in
the last 2 decades. One of the best studied of these so-called
hyperthermophiles is the anaerobic archaeon Pyrococcus
furiosus, which grows optimally at 100°C by the fermentation of
carbohydrates and peptides (15). The organism uses a
modified Embden-Meyerhof glycolytic pathway that contains several novel
enzymes (13, 23). For example, both hexokinase and
phosphofructokinase are ADP- rather than ATP-dependent enzymes
(24, 48). In addition, the expected
glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase
enzymes, which would convert glyceraldehyde-3-phosphate to
3-phosphoglycerate with the concomitant phosphorylation of ADP to ATP
and reduction of NAD+ to NADH, appear to be replaced by a
single enzyme, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase
(GAPOR). GAPOR oxidizes glyceraldehyde-3-phosphate directly to
3-phosphoglycerate and uses ferredoxin rather than NAD+ as the electron acceptor. Purified GAPOR is a unique
tungsten-containing enzyme that has no known analog in mesophilic
archaea or bacteria (31). Another unusual step in glucose
catabolism includes the conversion of acetyl coenzyme A (acetyl-CoA) to
acetate. In anaerobic bacteria, this is a two-step process via acetyl
phosphate catalyzed by phosphotransacetylase and acetate kinase. In
P. furiosus, however, these two enzymes are replaced by
acetyl-CoA synthetase, which converts acetyl-CoA directly to acetate
and phosphorylates ADP to ATP (29). Other enzymes involved
in glucose oxidation that have been purified from Pyrococcus
species include pyruvate:ferredoxin oxidoreductase (4),
enolase (36), and triosephosphate isomerase (25). In contrast to the enzymes described previously,
these are quite similar (except in thermostability) to their mesophilic counterparts.
P. furiosus also synthesizes glucose during growth on
peptide-derived amino acids (15), but less work has been
done to explore the gluconeogenic pathway. This is thought to occur by
a reversal of the conventional Embden-Meyerhof pathway, and the
activities of the enzymes proposed to be involved have been measured in
cell extracts (44). Of the gluconeogenic-specific enzymes,
only phosphoglycerate kinase (19) has been purified from
Pyrococcus species. Herein we focus on the enzyme that
carries out the first step in the conversion of pyruvate to glucose,
phosphoenolpyruvate (PEP) synthetase, which catalyzes the
phosphorylation of pyruvate according to equation 1:
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(1)
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Transcriptional analyses indicated that the cellular
concentration of PEP synthetase increased when a glycolytic substrate such as maltose was added to the growth medium of P. furiosus (38, 39). To explain this result, it was
suggested that this enzyme functions in glycolysis (reverse of equation
1) as well as in gluconeogenesis (39). However, there are
no kinetic data to support this contention. It was, therefore, of some
relevance to determine the catalytic properties of PEP synthetase from
this organism.
Surprisingly, little is known about PEP synthetases from prokaryotes,
in spite of their importance in controlling carbon flow during glucose
metabolism (20, 28, 37, 45, 47). The only one to be
studied extensively is that from Escherichia coli (10,
11, 32, 33, 34). It is a cofactorless homodimer with a molecular
mass of 150 kDa and catalyzes the phosphorylation of pyruvate by using
ATP to generate PEP, AMP, and phosphate. Only two other PEP synthetases
have been purified and examined to any extent, and both are from
archaea, the moderately thermophilic methanogen Methanobacterium
thermoautotrophicum (14) and the heterotrophic
hyperthermophile Staphylothermus marinus (8, 9, 16,
17). The quaternary structure of the former enzyme was not
reported, but S. marinus PEP synthetase exists as a large homomultimeric complex containing 24 subunits (17).
Conversely, while the kinetic properties of the latter enzyme are
unknown, those of the M. thermoautotrophicum enzyme are
similar to those of E. coli PEP synthetase
(14). It was therefore of some interest to investigate the
physical and catalytic properties of the enzyme from P. furiosus, especially as the enzyme has been reported to be
sensitive to inactivation by oxygen (44). For example, did the enzyme exist as a large complex, did it function in both
gluconeogenesis and glycolysis, did it utilize nucleotides other than
ATP as the phosphate donor for PEP synthesis, and what may be the cause
of its oxygen sensitivity?
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MATERIALS AND METHODS |
Enzyme assays.
The methods used to characterize P. furiosus PEP synthetase required discontinuous assays where
reaction substrates or products were measured at 23°C after catalytic
conversion at 80°C. One unit of PEP synthetase activity is equivalent
to 1 µmol min1 of product (PEP or phosphate) formed in
the forward reaction (equation 1) or of pyruvate formed in the reverse
reaction. To measure phosphate formation, the enzyme sample and 4 mM
pyruvate were incubated at 80°C in 1 ml of 10 mM MgCl2,
200 mM KCl, 50 mM
N-2-hydroxyethylpiperazine-N'-3-propanesulfonic
acid (EPPS) buffer (pH 8.4). ATP (4 mM) was added to start the
reaction, and 0.2 ml of 5 M H2SO4 was added
after 2 min to quench it. The amount of phosphate produced was measured
as described previously (18).
The method used to determine the amount of PEP formed was modified from
that reported previously (
14). The reaction was
carried
out as described above, and the residual pyruvate was
first converted
to lactate by lactate dehydrogenase (LDH) (see
equation 2 below).
Aliquots were added to a reaction mixture containing
2 ml of 20 mM
MgCl
2 and 40 µM NADH and 5 U of LDH in 100 mM EPPS
buffer
(pH 8.0) maintained at 23°C. The PEP concentration was
then
determined by adding 1 mM ADP and 5 U of pyruvate kinase
(PK; Sigma) to
this reaction mixture. The amount of NADH oxidized
by LDH was
determined by the decrease in absorption at 340 nm
as pyruvate formed
from PEP (see equation 3 below) is converted
to lactate (equation 2). A
molar absorbance of 6,220 M
1 · cm
1
was used for NADH.
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(2)
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(3)
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Pyruvate formation by PEP synthetase was measured using
a method based on the previously published assay (
14). The
1-ml
assay mixture contained the enzyme sample, 4 mM AMP, 10 mM
phosphate,
10 mM MgCl
2, and 100 mM KCl in 50 mM EPPS buffer
(pH 8.4) at 80°C.
The reaction was initiated by the addition of 4 mM
PEP (reverse
reaction of equation 1). After 2 min, the reaction mixture
was
placed on ice to quench the reaction. Samples of the reaction
mixture were transferred to cuvettes containing 40 µM NADH, 10
mM
MgCl
2, and 100 mM KCl in 50 mM EPPS buffer (pH 7.5) in a
final
volume of 2 ml. The pyruvate formed was measured by adding LDH
(5 U) and determining the amount of NADH oxidized (equation
2).
PEP synthetase was also examined for its ability to catalyze pyruvate
phosphate dikinase (PPDK) (equation 4) and PK (equation
5) reactions.
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(4)
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(5)
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The PPDK activity was measured by adding 10 mM phosphate
as substrate in the PEP formation assay. The PK activity was measured
with a modified pyruvate formation assay; AMP and phosphate were
replaced with ADP (4 mM) as the substrate. The effect of pH on
enzyme
activity was tested using the following buffers at a concentration
of
50 mM (at the indicated pH values): sodium citrate (pH 4.0
to 5.5),
2-(
N-morpholino)ethanesulfonic acid (MES) (pH 5.5 to
7.0),
morpholinepropanesulfonic acid (MOPS) (pH 7.0 to 8.0), EPPS
(pH 8.0 to
9.0), and 2-(
N-cyclohexylamino)ethanesulfonic acid
(CHES)
(pH 9.0 to 10.0).
Purification of PEP synthetase.
P. furiosus (DSM
3638) was grown with maltose as the carbon and energy source in a
600-liter fermentor, as described previously (6). All of
the purification steps were carried out at 23°C. The cell extract and
the first chromatography step were anoxic, since other enzymes of
interest that are oxygen sensitive were purified from the same cell
batch. The buffers for these steps were repeatedly degassed and flushed
with Ar, contained sodium dithionite (2 mM) and dithiothreitol (2 mM),
and were kept under positive Ar pressure. Thereafter, the purification
was performed aerobically. P. furiosus cells (200 g [wet
weight]) were suspended in 600 ml of 50 mM Tris buffer (pH 8.0) with
DNase I (0.5 µg/ml) and were stirred for 2 h at 37°C to
suspend and lyse the cells. The cytosolic portion of the cell extract
was separated from the membranes by centrifugation at 50,000 × g for 2 h. This was applied to a column (10 by 14 cm) of
DEAE Sepharose fast-flow (Pharmacia) equilibrated with 50 mM Tris
buffer (pH 8.0) by using a Fast Protein Liquid Chromatography system
(Pharmacia). The extract was diluted threefold with equilibration
buffer as it was loaded onto the column at a rate of 15 ml/min. PEP
synthetase did not bind to the column and was collected in the
pass-through. This was filtered through a 0.2-µm-pore-size filter
(Maxiculture Capsule; Fisher) and was applied to an ion exchange column
(5 by 35 cm) of quaternary ammonium (Q)-Sepharose fast-flow (Pharmacia)
equilibrated with 50 mM glycine buffer (pH 9.5). The filtered protein
was diluted fourfold with equilibration buffer and loaded onto the
column. The bound protein was eluted with a 0 to 0.5 M NaCl linear
gradient (3,500 ml) in 50 mM glycine (pH 9.5) using a flow rate of 5 ml/min. PEP synthetase activity was measured in fractions collected
from the column during the 0.38 to 0.43 M NaCl portion of the gradient. The fractions with specific activity above 7 U/mg were pooled and
concentrated by ultrafiltration (PM 30; Amicon). The concentrated sample was applied to a column (3.5 by 60 cm) of Superdex 200 (Pharmacia) equilibrated with 50 mM Tris buffer (pH 8.2) containing 0.2 M NaCl at 0.7 ml/min. PEP synthetase eluted in the void volume. Those
fractions judged to be pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were concentrated and stored in liquid
N2.
Other methods.
The molecular weight of the holoenzyme was
determined using an analytical Superose 6 (Pharmacia) gel filtration
column (1 by 28 cm) equilibrated with 50 mM Tris buffer (pH 8.4)
containing 0.2 M NaCl. The standards used to calibrate the column were
alcohol dehydrogenase (150 kDa), 
-amylase (200 kDa), catalase (223 kDa), apoferritin (440 kDa), thyroglobin (663 kDa), and human
immunoglobulin M (900 kDa). The subunit molecular weight was determined
by SDS-PAGE with an 8% acrylamide-Tris-glycine-buffered gel, as
described previously (26). The thermal stability of PEP
synthetase was determined by incubating samples at 90°C for various
time intervals in 50 mM EPPS buffer (pH 8.0) containing 0, 0.2, or 0.5 M KCl. The samples were placed on ice and were assayed immediately by pyruvate formation at 80°C. Metal content was determined by
inductively coupled plasma emission spectroscopy (ICP) on a Jarrel Ash
Plasma Comp 750 instrument at the Chemical Analysis Laboratory of the University of Georgia. The N-terminal amino acid sequence of the protein was determined (using an Applied Biosystems model 477 sequencer) by the Molecular Genetics Instrumentation Facility at the
University of Georgia. The PEP synthetase sample was prepared for
sequencing by electrophoresis on an SDS-15% acrylamide Tris-glycine gel followed by electroblotting (Bio-Rad blotting system) onto a
polyvinylidene difluoride sequencing membrane. The electroblotting was
carried out at 200 mA for 7 h in
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer (pH 11.0)
containing 10% (vol/vol) methanol. Protein concentrations were
estimated by the method described by Bradford (5).
Substrate requirements and kinetic constants of PEP synthetase were
determined using the phosphate formation assay for the
forward reaction
and the pyruvate formation assay for the reverse
reaction. The effects
of the reducing agents dithiothreitol (5
mM) and
-mercaptoethanol
(40 mM) were tested by incubating them
with the enzyme followed by
anaerobic PEP formation assay; vials
containing reaction mixture were
degassed and placed under Ar
with either dithiothreitol or
-mercaptoethanol. The specificity
of the PEP synthetase for
-keto
acids, phosphate donors, and
phosphate acceptors was determined by
using both the phosphate
formation and pyruvate formation assays. The
PEP formation assay
was used to study the effect of divalent and
monovalent cations
on enzyme activity. The calcium requirement of PEP
synthetase
was determined by first removing calcium by EGTA treatment.
The
enzyme (1 mg ml
1 in 50 mM Tris [pH 8.0]) was
incubated with 1 mM EGTA for 30 min
at 25°C or for 5 min at 80°C
and was then passed through a Sephadex
G-25 column (5 ml) equilibrated
with 50 mM Tris, pH 8.0. The metal
content was measured by ICP
analysis, and the catalytic activity
of the EGTA-treated enzyme was
determined by the phosphate formation
assay.
| |
RESULTS |
Purification of PEP synthetase.
Cell extracts of P. furiosus cells grown with maltose as the carbon source contained
high PEP synthetase activity. Values of 1.2 ± 0.5 U mg1
were obtained using the phosphate formation assay. The results of a
typical purification procedure are summarized in Table
1. The yield of the pure enzyme from 200 g (wet weight) of cells was approximately 50 mg with a 20-fold
purification factor, suggesting that PEP synthetase represents a
significant portion (~5%) of the cytoplasmic protein (Table 1). The
final specific activity of the protein that was 
90% homogeneous as
judged by SDS-PAGE (see below) was 18 ± 4 U mg1
using the phosphate formation assay. During the last steps of the
purification, the concentration of the PEP synthetase had to be
maintained below 10 mg ml1 (pH 7.5 to 8.5); otherwise the
solution became viscous and the protein precipitated. In catalyzing PEP
formation, the purified enzyme was dependent on both ATP and pyruvate
for activity and there was no increase in activity when phosphate was
added as an additional substrate. This result indicates that the enzyme is a PEP synthetase (equation 1) rather than a PPDK (equation 4). The
enzyme also catalyzed the reverse reaction (the formation of pyruvate
from PEP, AMP, and phosphate), and AMP and phosphate could not be
replaced by ADP, showing that the enzyme is not a PK (equation 5).
Physical properties.
During the final purification step, PEP
synthetase eluted in the void volume of a Superdex 200 gel filtration
column, indicating that it had a molecular mass of at least 600 kDa.
Subsequent chromatography of the purified protein on a column of
Superose 6 (with an exclusion limit of 40 MDa) gave rise to two protein
fractions. One eluted with an apparent molecular mass of 690 ± 20 kDa, while the other had a molecular mass of 1.64 ± 0.01 MDa. The
two forms were indistinguishable by SDS-PAGE analysis, and their
N-terminal amino acid sequences were identical (see below). The larger
form accounted for about 30% of the total protein applied to the
column. However, only the smaller of the two protein forms had PEP
synthetase activity; no activity (<0.01 U mg1 in the
phosphate release assay) was detected with the larger form. Removal of
the inactive 1.6-MDa form resulted in an increase in the specific
activity of the "pure" protein by 1.7-fold.
The purified protein gave rise after SDS-PAGE analysis to one band that
corresponded to a molecular mass near 92 kDa (Fig.
1). However, on some occasions, a second
band of comparable size
was observed with some enzyme samples. This
result is consistent
with the behavior of the
S. marinus
enzyme (8). The specific activities
of the various preparations (with
one or two bands) were comparable,
and the two forms had the same
N-terminal amino acid sequence
(AYRFIKGFEELSKNDVPLVG), showing
that they are different forms
of the same enzyme. These are thought to
be phosphorylated and
nonphosphorylated forms, as discussed below.
Their sequences closely
matched that previously deduced from the gene
sequence of
P. furiosus PEP synthetase (19 of 20 residues
[21, 39]), with the exception
of the encoded N-terminal methionine,
which was not present (
21).
The calculated molecular mass
of the subunit (without the N-terminal
methionine) from the gene
sequence is 90,346 Da, which is approximately
the size of the subunit
as determined by gel electrophoresis.
The active form of the enzyme
(690 kDa) therefore appears to be
a homooctomer. ICP spectroscopic
analysis of the pure protein
giving rise to a single major band on the
SDS gel revealed 1.1
± 0.40 g-atoms of Ca and 1.1 ± 0.43 g-atoms of phosphorus per
92,000 Da of protein. Metals such
as Zn, Co, and Cu were not present
in significant amounts (<0.1
g-atoms subunit
1). All preparations of PEP synthetase
examined contained iron
in amounts of 0.1 to 0.4 g-atoms
subunit
1. However, the enzyme specific activity did not
correlate with
the iron concentration, so it is assumed that this metal
is not
a functional part of the enzyme. The presence of phosphorus is
consistent with previous data on the PEP synthetase from
E. coli.
This enzyme can be isolated in a phosphorylated form, which
is
an intermediate in the enzyme's phosphate transfer mechanism
(
3),
although the
E. coli protein has not been
reported to contain
calcium. To determine if calcium was adventitiously
bound to
P. furiosus PEP synthetase, the enzyme was
incubated with EGTA at
23°C or at 80°C for 30 min and then
separated from the chelator
by ion-exchange chromatography (Pharmacia).
The enzyme did not
lose activity during either treatment, and ICP
analysis showed
that the calcium content remained the same (~1 g-atom
subunit
1). This element therefore appears to be an
integral part of the
P. furiosus PEP synthetase.
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FIG. 1.
SDS-PAGE of P. furiosus PEP synthetase. The
right lane contained purified PEP synthetase, and the left lane
contained marker proteins with molecular masses (in kDa) indicated at
the left.
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Previous researchers concluded that PEP synthetase might be redox
active, since the enzyme from
P. furiosus was oxygen
sensitive
(
44) and the enzyme from methanogens required
reducing agents
for maximum activity (
14). However, the
pure
P. furiosus enzyme
lost no activity when stored
overnight under air (or Ar), and
incubation for 30 min with either 5 mM
dithiothreitol or 40 mM
-mercaptoethanol, either at 23 or 80°C,
had no affect on its
activity. The thermal stability of the enzyme (1 mg ml
1 in 50 mM EPPS, pH 8.0) was measured at 90°C, and
the time required
to lose 50% of its activity
(
t1/2) was about 9 h. The presence
of KCl,
either at 0.2 or 0.5 M, had no effect on the
t1/2 value.
To investigate whether thermal
inactivation was due to a change
in the quaternary structure of the
enzyme, a sample (1 mg ml
1 in 50 mM Tris, pH 8.0) that
had been incubated at 90°C for 10
h was cooled on ice and then
applied to a Superose 6 gel filtration
column. The proportion of the
larger, inactive form of the enzyme
remained about the same (increasing
from 29 to 34% of the total),
indicating that aggregation of the
active 690-kDa form was not
the main cause of loss of enzyme
activity.
Catalytic properties.
PEP synthetase required
divalent metal cations in the assay mixture for catalysis as
measured by the PEP formation assay; no activity was detected if they
were omitted. The optimal Mg2+ concentration was 10 mM
(range tested, 0 to 50 mM). At Mg2+ concentrations above 10 mM, there was slight inhibition. The activity of the enzyme was also
measured in the presence of Ca2+, Sr2+,
Mn2+, Co2+, and Ni2+ (2 mM, as the
chloride salts) to see if they could substitute for Mg2+.
This was the case with Mn2+ (at 95% of Mg2+
activity) and Co2+ (93%) ions, while Ni2+
(15%) was not as effective and both Sr2+ (4%) and
Ca2+ (3%) showed only minor activation. The effects of the
monovalent cations Na+, K+, and
NH4+ (chloride salts) on PEP synthetase were
measured in the presence of 10 mM Mg2+ ions. Enzyme
activity was stimulated by the addition of NH4+
(48% increase with 100 mM) or K+ (50% with 100 mM) ions,
but Na+ (100 mM) had no effect. The potassium ion
concentration for optimal enzyme activity was between 30 and 250 mM,
within the intracellular range (30). The enzyme showed an
optimal pH near 9.0 in the PEP formation assay, while the optimal
concentration of the pyruvate formation reaction was 7.5 (Fig.
2). The optimal temperature for PEP
formation over a 2-min period was 90°C. The enzyme exhibited measurable activity at 30°C, but it was only 0.2% of that measured at 90°C (data not shown).
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FIG. 2.
pH dependence of the pyruvate (empty symbols) and PEP
(solid symbols) formation reactions of P. furiosus PEP
synthetase. The enzyme activities were determined under the conditions
described in Materials and Methods but at the indicated pH.
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Kinetic constants were determined for pyruvate and ATP in the forward
reaction and for PEP, AMP, and phosphate in the reverse
reaction. The
substrate concentrations of the invariant substrates
were 4 mM except
in the case of phosphate, which was used at 10
mM. The variable
substrates were used at 0.004 to 4 mM in all
cases except for
phosphate, where the substrate concentration
varied between 0.1 and 100 mM. As shown in Table
2, the enzyme
catalyzed PEP formation with a catalytic efficiency that is at
least
100-fold greater than that measured for the reverse reaction,
consistent with the proposed physiological role of the enzyme
in
gluconeogenesis. The specificity of PEP synthetase was examined
with a number of 2-ketoacids in addition to pyruvate, and these
included 2-keto-3-methylvalerate, 2-ketoisocaproate,
2-ketoisovalerate,
2-ketocaproate, 2-ketobutyrate,
2-ketoglutarate, 2-ketomalonate,
phenylpyruvate,
hydroxyphenylpyruvate, imidazolpyruvate, and indole-3-pyruvate.
The enzyme has a strong preference for pyruvate and only a slight
amount of phosphorylation of 2-ketobutyrate (2 mM, showing 8%
of the
activity measured with pyruvate) and 2-ketoglutarate (2
mM, 5.3%). The
substrate specificity is reasonable since it is
not clear what the cell
would do with phosphorylated forms of
butyrate and glutarate.
PEP synthetase was tested for the ability to use alternate phosphate
donors and acceptors, since there is a precedent for
unusual nucleotide
usage of enzymes in
P. furiosus (
24,
29,
48).
Of the phosphate donors tried, ATP was the only substrate
that the
enzyme would use to catalyze PEP formation. The activity
with ADP, GDP,
or GTP (each at 2 mM) was less than 1% of the activity
of the enzyme
with ATP. In the reverse direction, ADP, AMP, GDP,
and GMP (2 mM), with
or without phosphate, were tested as phosphate
acceptors for PEP
synthetase. The enzyme had a strong preference
for AMP with phosphate
as a substrate, although there was significant
activity (20% of that
with AMP) using ADP with
phosphate.
| |
DISCUSSION |
Unlike several other nucleotide-utilizing enzymes recently
purified from P. furiosus, such as hexokinase (glucokinase),
phosphofructokinase, and acetyl-CoA synthetase (24, 29,
48), PEP synthetase is typical in that it uses the nucleotides
expected from studies of gluconeogenesis in mesophilic organisms.
Pyruvate and ATP are used to generate PEP, AMP, and Pi, and
catalytic efficiencies suggest that the reaction occurs in the
gluconeogenic direction. The enzyme does not catalyze to any extent
PPDK (equation 4) or PK (equation 5) reactions. However, the enzyme was
unique relative to its mesophilic counterparts in that it was a
homooctomer and comprised a large proportion (~5%) of the total
cytosolic protein.
Little is known about PEP synthetases in general, so there is only
limited information with which to compare the properties of the
P. furiosus enzyme (Table 3).
The most obvious difference between those purified thus far is size.
The homomultimeric enzymes from the hyperthermophilic archaea S. marinus and P. furiosus are much larger than the
homodimeric E. coli protein. Elegant and extensive
structural studies on S. marinus PEP synthetase by electron
microscopy have shown that it contains 24 subunits. In contrast, the
P. furiosus enzyme is purified in what appear to be 8- and
18-subunit forms, with only the homooctomer possessing enzymatic
activity. Unfortunately, whether S. marinus PEP synthetase is active has not been reported, as it was purified according to its
size (8), and it is not known whether this organism contains a smaller (and active) version of the enzyme. It has been
suggested that the increase in the number of subunits of hyperthermophilic enzymes compared to that for their mesophilic analogs
might contribute to their increased thermostability (2, 12, 27,
50). Whether the larger and seemingly inactive version of
P. furiosus PEP synthetase is an artifact of purification or whether it has a physiological significance remains to be determined.
Studies on the response of growing P. furiosus on maltose
showed that maltose addition caused an increase in the concentration of
the mRNA encoding PEP synthetase (38, 39). However, PEP synthetase activity varied less than twofold in cultures grown on
maltose versus on peptides (1). To explain the first
result, it was suggested that this enzyme not only played a role in
gluconeogenesis but also catalyzed the conversion of PEP to pyruvate in
the glycolytic pathway of P. furiosus, a conversion normally
catalyzed by PK (38, 39). However, the kinetic properties
of the P. furiosus PEP synthetase (Table 2) indicate that
the gluconeogenic reaction is greatly favored and that the enzyme has
extremely low PK activity in comparison.
There is some confusion about which enzyme catalyzes the conversion of
PEP to pyruvate in P. furiosus. It was recently suggested that the organism contains a novel AMP-dependent PK that forms pyruvate
and ATP from PEP, AMP, and Pi, the reverse reaction of PEP
synthetase (42). This is highly significant, as such an enzyme would have the effect of regenerating ATP from the AMP formed by
the ADP-dependent kinases in the earlier steps of the glycolytic
pathway. However, the genome sequence of P. furiosus (http://comb5-156.umbi.umd.edu/genemate) contains an open reading frame that is homologous to conventional PKs (e.g., it shows 48% similarity to the amino acid sequence of PK from Bacillus
stearothermophilus), and cell extracts of P. furiosus
contain significant ADP-dependent PK activity (1.4 U mg1
at 100°C [43]). Interestingly, the novel AMP-dependent PK exhibits Km values for PEP, AMP, and Pi not
too dissimilar from those reported here for PEP synthetase (although
they were measured at pH 6.5 and 50°C [42]). Moreover, although the
subunit size of this novel PK (78 kDa) is much smaller than that of PEP
synthetase (92 kDa by SDS-PAGE; 90.3 kDa from the sequence), its
N-terminal amino acid sequence reportedly (T. Ohshima, H. Sakuraba,
H. J. Schreier, E. Utsumi, and N. Nunoura-Kominato, Abstr. 3rd
Int. Congr. Extremophiles, abstr. L30, 2000) matches that of the PEP
synthetase described herein, an enzyme whose complete amino
acid sequence is homologous to those for other known PEP synthetases
(21). Thus, while the true nature of the novel PK
(42) is unclear, the results presented herein are fully
consistent with PEP conversion to pyruvate and pyruvate conversion to
PEP occurring in P. furiosus through conventional PK and PEP
synthetase reactions, respectively. In addition, extracts of P. furiosus also contain significant adenylate kinase activity (0.22 to 0.37 U mg1 [1]), suggesting that the interconversion
of AMP, ADP, and ATP also occurs by a conventional mechanism.
What reasons are there, then, for such a relatively large amount of PEP
synthetase in P. furiosus (~5% of the cellular protein)? Under such conditions it seems unreasonable that the PEP produced by
the PEP synthetase reaction would be required for biosynthesis, since sufficient PEP should be formed in the glycolytic pathway. One possible explanation is that a futile cycle between PEP and pyruvate exists in this organism when grown under our conditions. It
has been suggested that such futile cycles can be used by some bacteria
when they are grown in the presence of high carbohydrate concentrations
to remove excess energy, as this can be harmful to the cell
(40). Such organisms experience an imbalance in metabolism, as the energy produced from carbohydrate oxidation via
glycolysis is much greater than the energy required for cell maintenance and growth (41, 51). The disposal of the
catabolic energy generated in excess of the cells' anabolic needs has
been termed "energy spilling" (41). The P. furiosus cells used in the current study were grown in a medium
with a high maltose concentration (6), so the cells could
well experience the metabolic imbalance that makes energy spilling
advantageous. However, futile cycling between PEP and pyruvate via PEP
synthetase has not yet been demonstrated in any organism. In E. coli, for example, such cycling between glycolytic and
gluconeogenic enzymes is prevented by the tight regulation of
these pathways (7, 35). However, regulation in
P. furiosus seems to occur only at the level of
glyceraldehyde-3-phosphate, via glyceraldehyde-3-phosphate
dehydrogenase and the novel enzyme GAPOR (48, 49).
Therefore, it is possible that PEP synthetase could be functioning in
P. furiosus as an energy-spilling mechanism. Growth studies
similar to those reported previously (22) but using an
extended range of carbohydrate concentrations are required to determine
whether this is the case and to see whether differences in growth yield
accompany changes in PEP synthetase activity. Such studies are underway
(38, 39). Whether regulation also involves the
interconversion between the smaller, active and larger, inactive forms
remains to be established.
| |
ACKNOWLEDGMENTS |
We thank Marc Verhagen for helpful discussions and suggestions.
This research was supported by grants from the National Science
Foundation (MCB 9809060 and BES-0004257).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Life Sciences Building, University of Georgia, Athens, GA
30602. Phone: (706) 542-2060. Fax: (706) 542-0229. E-mail: adams{at}bmb.uga.edu.
| |
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Journal of Bacteriology, January 2001, p. 709-715, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.709-715.2001
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Abstract
Introduction
