(Received for publication, August 30, 1994; and in revised form, November 18, 1994)
From the
The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP. Whereas E. coli PFK (EcPFK) is profoundly inhibited by ATP, B.
stearothermophilus PFK (BsPFK) is only slightly inhibited. The
structural basis for this difference could be closure of the active
site via a conformational transition that occurs in the ATP-binding
domain of EcPFK, but is absent in BsPFK. To investigate the role of
this transition in ATP inhibition of EcPFK, we have constructed a
chimeric enzyme that contains the ``rigid'' ATP-binding
domain of BsPFK grafted onto the remainder of the EcPFK subunit. The
chimeric PFK has the following characteristics: (i) tetrameric
structure and kinetic parameters similar to those of the native
enzymes, (ii) insensitivity to regulation by the effector
phosphoenolpyruvate despite its ability to bind to the enzyme, and
(iii) a sigmoidal (n around 2) fructose
6-phosphate saturation curve. From the results, it is concluded that
the active site regions of the two native enzymes are remarkably
similar, but their effector sites and their mechanisms of heterotropic
regulation are different. The chimeric subunit is locked in a structure
resembling that of activated E. coli PFK, yet the enzyme can
exist in two different conformational states. Mechanisms for its
sigmoidal kinetics are discussed.
Phosphofructokinase (EC 2.7.1.11) (PFK) ()catalyzes
the transfer of the
-phosphate from MgATP to fructose 6-phosphate
(Fru-6P) in the first committed step of glycolysis. In bacteria, PFK is
regulated heterotropically by the activator ADP (or GDP) and the
inhibitor phosphoenolpyruvate (PEP). The PFKs from the bacteria Escherichia coli and Bacillus stearothermophilus are
remarkably similar in structure (Evans et al., 1981;
Shirakihara and Evans, 1988). The enzymes are both tetramers of
identical subunits, their subunit
-carbon traces are nearly
superimposable, and they share 55% amino acid identity. They can be
viewed as dimers of rigid dimers. In each enzyme, the subunit is
divided into a large and a small domain, with the active site located
in a cleft between the two domains. There are thus four active sites.
There are also four effector sites into which PEP and ADP (or GDP)
bind; these are located in deep clefts between subunits of the rigid
dimer. Within the active site, the amino acid residues that bind ATP
are almost entirely from the large domain, while those that bind Fru-6P
are mostly from the small domain, but include 2 arginines (arginines
162 and 243) from across the dimer-dimer interface. Coordination of the
Mg
ion as well as transfer of the
-phosphate of
ATP involves residues from both domains.
Despite the remarkable structural similarity between E. coli PFK (EcPFK) and B. stearothermophilus PFK (BsPFK), there is a significant kinetic and allosteric difference between them. Whereas Fru-6P saturation of BsPFK is hyperbolic in the presence of saturating MgATP levels (Valdez et al., 1989), Fru-6P saturation of EcPFK is highly cooperative (Hill number around 4.0) under the same conditions (Blangy et al., 1968). ATP has been termed an ``allosteric'' inhibitor of EcPFK (Evans, 1992) because of its ability to profoundly inhibit the enzyme at low Fru-6P concentration (Kundrot and Evans, 1991). On the other hand, ATP only slightly inhibits BsPFK, and the inhibition is clearly non-allosteric. Blangy et al.(1968) have shown that EcPFK obeys the Monod-Wyman-Changeux model of allosteric behavior (Monod et al., 1965), in which the protein exists in an equilibrium involving two conformational states, a low activity T state and a high activity R state. The allosteric behavior of EcPFK has been explained in terms of this model. Recent studies, however, suggest that the MWC model cannot fully account for the cooperativity of EcPFK (Deville-Bonne et al., 1991a; Berger and Evans, 1992).
X-ray diffraction studies indicate that two different subunit
structures are present in the EcPFK tetramer, ``open'' and
``closed'' (Shirakihara and Evans, 1988). Therefore, an
open-to-closed transition may be taking place within the EcPFK subunit.
A comparison between the -carbon traces of the open and closed
subunit structures reveals that most of the movement occurs within a
region of the large (ATP-binding) domain. The transition apparently
does not occur within the large domain of BsPFK (Schirmer and Evans,
1990).
We have constructed and studied a chimeric PFK, composed of parts of BsPFK and EcPFK, in order to investigate the structural basis for their different regulation by ATP. The chimeric enzyme (ChiPFK) contains a portion of the ``rigid'' large domain of BsPFK (the ATP-binding domain) grafted in-frame onto the remainder of the EcPFK subunit. The active site of ChiPFK is thus composite: residues that bind ATP are from BsPFK while those that bind Fru-6P are from EcPFK. Steady-state kinetics and fluorescence studies were performed on the chimeric PFK and the two native enzymes. The results indicate that the active site of ChiPFK is locked in an open conformation that resembles that of the activated form of EcPFK. Nevertheless, the enzyme displays sigmoidal Fru-6P saturation kinetics (Hill number 1.7 ± 0.2). The absence of regulation by PEP despite its ability to bind in the effector site indicates that the structural pathways of allosteric PEP inhibition are different between the two native enzymes. In many respects, ChiPFK resembles a proteolyzed derivative of EcPFK (Le Bras and Garel, 1982) that is cooperative yet insensitive to allosteric effectors. The possible origins of ChiPFK sigmoidal saturation kinetics are discussed.
Primer 1 was designed to hybridize
to pUC18 itself just beyond the EcoRI cloning site, which is
located upstream of the bspfk promoter in recombinant bspfk/pUC18, and located downstream of the ecpfk termination sequence in recombinant ecpfk/pUC18. In both
cases, primer 1 points into the pfk insert. Primer 2 was
designed to hybridize to the bspfk coding strand with its 3`
end located at the codon for Pro, pointing upstream.
Primer 2 contains an internal ApaI restriction site
(underlined above) that also causes the mutation
Val
Ala. (
)Primer 3 was designed to
hybridize to the ecpfk complementary strand with its 3` end
located at the codon for Ile
, pointing downstream. Like
primer 2, it contains an internal ApaI site. PCR using primers
1 and 2 amplifies a region of bspfk stretching from beyond its
promoter to codon 125. PCR using primers 1 and 3 amplifies a region of ecpfk stretching from codon 118 to beyond its termination
sequence. PCR was performed using a Perkin Elmer-Cetus DNA thermal
cycler, a GeneAmp
PCR kit, and AmpliTaq
DNA polymerase. The procedure suggested in the kit was followed.
The chimeric gene was created by digesting the two PCR products with ApaI, then ligating the fragments together with T4 DNA ligase.
In this way, the BsPFK gene up to codon 122 was grafted in-frame onto
the EcPFK gene beginning at codon 123. The chimeric gene was cloned
into pUC18 via its HindIII and EcoRI restriction
sites. The integrity of the chimeric gene was verified by directly
sequencing the entire coding region using a Sequenase kit
(U. S. Biochemical Corp., Cleveland, OH). No unintentional mutations
were found.
Expression of ChiPFK was performed as follows: recombinant chipfk/pUC18 plasmid DNA was transformed into competent DF1020 cells. The cells were plated onto Luria broth (LB) agar containing 50 µg/ml ampicillin, and the plates were incubated overnight at 30 °C. Six transformants were picked, directly inoculated into 3-ml volumes of LB-ampicillin (50 µg/ml) medium, and grown at 30 °C with agitation. The cultures reached stationary phase after about 60 h. A 1-ml aliquot of the culture having the highest PFK activity was inoculated into 500 ml of LB-ampicillin (50 µg/ml). This large culture was grown at 30 °C, reaching stationary phase after about 24 h. The cells were harvested by centrifugation at 4 °C. Cell pellets were brought up in buffer A (50 mM Tris-Cl, pH 7.9, 1 mM EDTA and 8 mM DTT) containing 1 mM phenylmethylsulfonyl fluoride, frozen in liquid nitrogen, and stored at -20 °C until ready for use.
The frozen cell suspension was thawed, then
sonicated, and centrifuged. The resulting clear supernatant was loaded
onto a Cibacron Blue 3GA-agarose column that had been equilibrated with
buffer A. The loaded column was washed with 10 volumes of buffer A, and
ChiPFK was then eluted with 2 mM ATP, 10 mM MgCl in buffer A. (The column was not washed with salt
prior to elution with ATP because NaCl concentrations as low as 100
mM caused elution of ChiPFK.) The most active fractions were
pooled and dialyzed against buffer A to remove the ATP and
Mg
. The column was regenerated by washing it first
with several volumes of 1.5 M NaCl, then extensively with
buffer A. The dialyzed pool containing PFK was then reloaded, the
column washed with 5 volumes of buffer A, and ChiPFK eluted with 0.5
mM Fru-6P in buffer A. PFK activity came off in a broad peak.
Fractions having the highest activity were pooled. The pool was either
1) concentrated using an Amicon (Beverly, MA) pressure cell, then
stored at 4 °C as a 55% ammonium sulfate suspension, or 2) dialyzed
in buffer A to remove the Fru-6P, then concentrated in 50% glycerol,
50% buffer A containing 2 mM ATP/10 mM MgCl
, and stored at -20 °C. The enzyme was
shown to be pure by electrophoresis on a 12% SDS-polyacrylamide gel.
Steady-state fluorescence measurements were made
at 25 °C using either a SPEX 1680 or an SLM 8000C fluorescence
spectrometer. The excitation source was a Xenon arc lamp. Intrinsic
fluorescence emission due to the single tryptophan, Trp,
of EcPFK or ChiPFK was measured at 340 nm following excitation at 295
nm. Slit widths were set at 8 nm. Protein concentrations were
5-25 µg/ml (below 1 µM), which is low enough to
avoid the inner-filter effect. The enzyme was buffered in 100 mM Tris-Cl, pH 8.2, containing 10 mM MgCl
, 5
mM NH
Cl, 0.25 mM EDTA, and 2 mM DTT. Two types of experiments were performed: 1) addition of a
saturating amount of ligand, and 2) addition of increments of ligand to
the enzyme solution. Corrections were made to compensate for volume
change, enzyme dilution, and nonspecific quenching. This was done by
performing parallel experiments in which phosphate of the same
concentration was added to the enzyme.
Figure 3:
Dependence of PFK activity on MgATP
concentration when Fru6P concentration is low (50 µM). A, initial velocity (units/µg) versus MgATP
concentration for native EcPFK in the absence of activator GDP. The inset shows the data between 0.1 and 4 µM MgATP
fit to the Michaelis-Menten equation. B, relative activity (v/V) versus MgATP
concentration for native E. coli PFK fully activated with 2
mM GDP (
) and chimeric PFK (
) (no GDP present). The
curve for BsPFK (dashed line) is included for comparison. The
data were fit to the equation for a steady-state random kinetic
mechanism (Ferdinand, 1966). For the assays at 10 mM MgATP,
the MgCl
concentration was 20
mM.
Figure 1:
Schematic diagram of the chimeric PFK
subunit. NH terminus is at the left. The closed bar represents the portion from B.
stearothermophilus PFK, while the open bar represents the
portion from E. coli PFK. The junction is at residue 122. Solid arrows indicate positions of residues that bind ATP, open arrows point to positions of residues that bind Fru-6P,
and the hatched arrow indicates the position of the lone
tryptophan.
Figure 2:
Dependence of PFK activity on Fru-6P
concentration. , native B. stearothermophilus PFK;
, native E. coli PFK;
, chimeric PFK. The inset shows the chimeric PFK plot on an expanded x axis. The MgATP concentration was saturating at 1.0
mM.
Figure 4:
Effect of PEP and GDP on PFK activity.
, B. stearothermophilus PFK;
, E. coli PFK;
, chimeric PFK. A, effect of PEP. Percent
activity remaining versus PEP concentration. [MgATP]
was saturating at 1.0 mM. [Fru-6P] was 0.3 mM for BsPFK and ChiPFK, and 1.5 mM for EcPFK. B,
effect of GDP. Percent activation versus GDP
concentration. [MgATP] was saturating at 1.0 mM.
[Fru-6P] was equal to the S
value,
which was 0.03 mM for ChiPFK and BsPFK, and 0.45 mM for EcPFK.
Figure 5:
Protection of native E. coli PFK (A) and chimeric PFK (B) against thermal
inactivation. Enzymes were incubated in 100 mM Tris-Cl, pH
8.2, containing 10 mM MgCl and 2 mM DTT
(at 60 and 50 °C for EcPFK and ChiPFK, respectively) in the
presence of no ligand (
), 5 mM Fru-6P (
), 10 mM PEP (
), or 5 mM GDP (
) for the indicated
time period. An aliquot was then removed and assayed for PFK activity.
MgATP and Fru-6P concentrations were both 1.0 mM in the
activity assays.
Figure 6:
Effect of AMPPNP on the Fru-6P-dependent
cooperative behavior of chimeric PFK. Plot of Hill number () or S
value (
) versus AMPPNP
Concentration. The Hill numbers and S
values
were obtained by fitting Fru-6P saturation data, collected in the
presence of 0.93 mM MgATP and the indicated
[AMPPNP], to the Hill equation. Error bars represent
standard errors.
We report studies on a chimeric bacterial phosphofructokinase
(ChiPFK), which has been successfully purified after high level
expression in PFK-deficient E. coli cells. ChiPFK exists as an
active tetramer similar in size to tetramers of the native enzymes from
which it is derived. Its catalytic rate constant is about half that of
either of the two native enzymes. Its affinities for substrates, as
measured by the K and S
values, are
similar to those of BsPFK and the activated form of EcPFK. Although
hybrid EcPFK tetramers have been studied before (Lau and Fersht, 1989),
this is the first time a chimeric PFK, containing parts of two
different PFKs grafted together, has been constructed and studied. Our
results attest to the great structural and functional similarity
between BsPFK and EcPFK, especially at their active sites. However, the
results also emphasize differences in their mechanisms of heterotropic
regulation.
Although stable as a tetramer in the sucrose gradient
sedimentation experiment, ChiPFK is somewhat unstable under the
conditions of the activity assay. The loss of ChiPFK activity over time
is paralleled by a quenching of its Trp fluorescence in
titration experiments. The fact that similar quenching profiles were
observed in the titration experiments irrespective of the ligand added
suggests that the effect is time- rather than ligand-dependent. It most
likely reflects dissociation of the active tetramer when present in
dilute solutions. This dissociation is to monomers rather than dimers
since quenching of Trp
fluorescence occurs when the EcPFK
tetramer dissociates into monomers, but not dimers (Deville-Bonne et al., 1989; LeBras et al., 1989).
Phosphoenolpyruvate does not inhibit ChiPFK. PEP binds to the
enzyme, but the binding causes no measurable change in Trp intrinsic fluorescence. This suggests that conformational changes
normally associated with allosteric inhibition are absent in ChiPFK.
The observation that GDP inhibits ChiPFK noncompetitively with respect
to MgATP indicates that GDP binds to ChiPFK. This binding may occur in
both the active and effector sites. The inability of GDP to protect
ChiPFK against thermal inactivation suggests that GDP does not bind the
effector site. However, GDP binding in the active site of BsPFK has
been shown to destabilize BsPFK in thermal inactivation
experiments.
Thus, such binding in the active site of
ChiPFK could destabilize the enzyme and mask the stabilization offered
by its binding in the effector site. The inability of GDP to induce a
fluorescence decrease in ChiPFK could thus be due either to poor
binding in the effector site or to an inability of the ChiPFK tetramer
to transmit allosteric changes. A third possibility is that the ChiPFK
tetramer is already locked in an activated state and cannot be further
activated by GDP.
The active site of EcPFK appears to close when AMPPNP binds to it, while the active site of ChiPFK is locked in an open conformation. EcPFK bound tightly to the Cibacron Blue affinity column during purification, and AMPPNP strongly inhibited subsequent Fru-6P binding to EcPFK in fluorescence studies. The latter result suggests that closure of the active site when AMPPNP binds in the absence of Fru-6P blocks subsequent binding of Fru-6P. Unlike EcPFK, ChiPFK associated loosely with the column during purification, and Fru-6P could bind to and cause a fluorescence decrease in ChiPFK after the enzyme had bound AMPPNP (20 µM). The active site of ChiPFK is thus locked open. Our results also suggest that the ChiPFK active site resembles that of the activated form of EcPFK. This is shown by similarities between ChiPFK and activated-EcPFK in their kinetic parameters (Table 1), their saturation by MgATP in the presence of low Fru-6P (Fig. 3B), and their AMPPNP versus Fru-6P inhibition patterns. Thus, the active site of ChiPFK is locked in an open structure similar to that of the activated form of EcPFK.
The results in Table 3indicate that ChiPFK can exist in two conformational states: a high fluorescence state induced by the binding of ATP or AMPPNP and a low fluorescence state induced by Fru-6P binding. The smaller fluorescence decrease seen when Fru-6P binds to ChiPFK compared to EcPFK (8 versus 19%, respectively) suggests that the conformational change is less, and perhaps different, in ChiPFK. The balance between high and low fluorescence conformational states depends on the relative amounts of AMPPNP and Fru-6P present. Furthermore, AMPPNP is more potent than Fru-6P in its ability to reverse the fluorescence state (low versus high) of the ChiPFK molecule. These binding results parallel the kinetic results showing that the relative amounts of Fru-6P and ATP present determine the reaction rate.
There are at least three possible explanations for the sigmoidal Fru-6P saturation kinetics of ChiPFK: 1) the sigmoidicity is due to apparent cooperativity arising from the kinetic mechanism. Both of the native enzymes from which ChiPFK is derived obey a steady-state random Bi Bi kinetic mechanism (Byrnes et al., 1994; Zheng and Kemp, 1992). This mechanism can allow two kinetically distinct pathways and sigmoidal saturation curves (Ferdinand, 1966). Although we have not rigorously determined the kinetic mechanism of ChiPFK, our results suggest that ChiPFK also obeys a steady-state random mechanism. 2) A slow isomerization occurs in the active site when Fru-6P binds in the presence of ATP. If the isomerization is slower than the catalytic rate, cooperativity can result (Ainslie et al., 1972). 3) Only two of the four active sites of ChiPFK are allosterically linked by a cooperative structural transition (Monod et al., 1965) that is triggered by interactions between ATP and Fru-6P in the active site.
The fact that two conformational states exist for ChiPFK casts doubt on the notion that its cooperativity is entirely kinetic in origin. Presumably, the ChiPFK molecule goes through a conformational transition as it moves from one state to the other according to the balance of substrate concentrations. Regarding this transition, the questions are: 1) is it a rapid and concerted one, involving more than one active site? or 2) is it slow relative to catalysis? A determination of whether the conformational transition is fast or slow relative to catalysis will have to await stopped-flow fluorescence measurements. However, whether fast or slow, the observation that ChiPFK is locked in an open conformation suggests that the transition almost certainly is not associated with closure of the active site.
The mechanism by which
AMPPNP abolishes the cooperativity of ChiPFK (Fig. 6) is not
known. However, it could be related to an interaction between the
imidophosphate group of AMPPNP and Arg, an active site
residue important in the cooperative behavior of EcPFK (Berger and
Evans, 1990). Arg
is involved in neutralizing the negative
charge on the transferred
-phosphate of ATP in the transition
state of EcPFK (Zheng and Kemp, 1994). The imidophosphate group of
AMPPNP is less acidic than the
-phosphate of ATP (Yount et
al., 1971). As a result, at pH 8.2 the
Arg
-imidophosphate interaction may be weakened relative to
the Arg
-phosphate interaction. A strengthening of the
interaction with Arg
has been proposed to occur when the
ATP analog adenosine 5`-[
-thio]triphosphate (ATP
S)
is used instead of ATP, in this case by perturbation of an interaction
with Thr
(Auzat et al., 1994). Whether the
conformational transition associated with ChiPFK cooperativity is
concerted or involves slow isomerization, the altered interaction with
Arg
could be important in explaining the effect of AMPPNP
on ChiPFK cooperative behavior.
In conclusion, we have found that the active site of the chimeric enzyme is locked in an open conformation similar to that of the activated form of E. coli PFK. Presumably, a conformational change responsible for closure of the active site has been disrupted in the chimeric enzyme, whose ATP-binding domain is from BsPFK. Yet, despite the ``openness'' of its active site, ChiPFK exhibits sigmoidal kinetics with respect to Fru-6P. We propose that the lower cooperativity of ChiPFK relative to EcPFK is related to its inability to undergo the open-to-closed transition involving its ATP-binding domain. Furthermore, the fact that some cooperativity remains in ChiPFK suggests that mechanisms in addition to one involving closure and opening of the active site are important for E. coli PFK cooperative behavior. Indeed, the complete mechanism for allosteric regulation of E. coli PFK is probably complex, involving binding cooperativity (Blangy et al., 1968), cooerativity due to slow transitions (Deville-Bonne et al., 1991a), and kinetic cooperativity arising from the kinetic mechanism (Zheng and Kemp, 1992).
Dedicated to the memory of David W. H. Chang(1971-1991).
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