©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Chimeric Bacterial Phosphofructokinase Exhibits Cooperativity in the Absence of Heterotropic Regulation (*)

(Received for publication, August 30, 1994; and in revised form, November 18, 1994)

W. Malcolm Byrnes (§) Wenjue Hu (¶) Ezzat S. Younathan (**) Simon H. Chang (**)

From the Department of Biochemistry, Louisiana State University, Baton Rouge, Louisiana 70803

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(H) 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.


INTRODUCTION

Phosphofructokinase (EC 2.7.1.11) (PFK) (^1)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 alpha-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 alpha-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.


EXPERIMENTAL PROCEDURES

Construction of the Chimeric Gene: PCR, Cloning, and Sequencing

The chimeric gene was constructed by ligating together two PCR amplification products: one from bspfk, the other from ecpfk. The two PFK genes (French and Chang, 1987; Hellinga and Evans, 1985) had previously been cloned into pUC18 plasmids and were oriented in opposite directions. Three oligonucleotide primers were used in the PCR amplification: primer 1, 5`-AGGAAACAGCTATGACCATGATTAC-3`; primer 2, 5`-CGTCCCCGGGGCCCCGACGCACG-3`; primer 3, 5`-CGTGCATCGGGGCCCCGGGCACTATC-3`.

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 ValAla. (^2)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 and Purification of PFKs

Both the chimeric PFK and the two native PFKs were expressed in PFK-deficient E. coli cells (DF1020 cells) that had been transformed with recombinant pUC18 plasmids containing the PFK genes. BsPFK was purified as described by Valdez et al.(1989), and EcPFK was purified as described by Kundrot and Evans(1991), but on a 40-fold larger scale. The enzymes were shown to be pure by electrophoresis on a 12% SDS-polyacrylamide gel using the method of Laemmli(1970).

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(2) 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(2), and stored at -20 °C. The enzyme was shown to be pure by electrophoresis on a 12% SDS-polyacrylamide gel.

Sedimentation Studies

Sucrose gradient sedimentation was performed according to the method of Martin and Ames(1961). Each 5-20% sucrose gradient was buffered with 100 mM Tris-Cl, pH 8.2, containing 10 mM MgCl(2), 5 mM NH(4)Cl, 0.25 mM EDTA, 2.0 mM DTT, and 0.5 mM Fru-6P (buffer B). An aliquot of a stock solution of PFK containing 2-5 µg was dialyzed at 4 °C against buffer B containing 2% sucrose, combined with a similarly dialyzed solution of yeast alcohol dehydrogenase (10 µg), then layered between the gradient (about 4 ml) and a volume of buffer B (about 1.4 ml) placed over the gradient. The alcohol dehydrogenase was used as an internal reference standard for determining the molecular weight of the sedimenting PFK species. Sedimentation was performed at 4 °C in a Beckman SW 50.1 rotor spun at 44,000 revolutions/min for 13 h. After sedimentation, fractions were collected by puncturing the bottom of the tube, then analyzed for PFK activity as described below. Fractions were analyzed for alcohol dehydrogenase activity by measuring the increase in absorbance at 340 nm in 1-ml assays that contained 100 mM Tris-Cl, pH 8.6, 1 mM NAD, and 8.4 µmol (50 µl) of ethanol.

Enzyme Activity Assays

Initial velocities were measured at 30 °C in 100 mM Tris-Cl, pH 8.2, containing 10 mM MgCl(2) and 5 mM NH(4)Cl by coupling the production of fructose 1,6-bisphosphate to the oxidation of NADH (0.2 mM). The coupled assay (Kotlarz and Buc, 1982) utilized the auxiliary enzymes aldolase (20 µg/ml), triosephosphate isomerase (10 µg/ml), and alpha-glycerophosphate dehydrogenase (10 µg/ml). Auxiliary enzymes were dialyzed at 4 °C against 100 mM Tris-Cl, pH 8.2, prior to use. ADP produced in the EcPFK-catalyzed reaction was regenerated to ATP using creatine phosphate (1 mM) and creatine kinase (10 µg/ml). However, this regenerating system was found to be unnecessary for the BsPFK- and ChiPFK-catalyzed reactions. Assays were initiated by addition of PFK. The change in absorbance at 340 nm was measured for at least 1 min following an initial nonlinear phase. A thermostatted Hitachi UV-2000 spectrophotometer was used for the measurements.

Fluorescence Measurements

The binding of various substrates or effectors can either enhance or quench the intrinsic fluorescence of the single tryptophan, Trp, of EcPFK (Berger and Evans, 1991). Trp is located near the C terminus of the EcPFK subunit within the large subunit-subunit interface of the rigid dimer. ChiPFK also has a tryptophan at position 311. (The intrinsic fluorescence of the single tryptophan of BsPFK, Trp, is largely insensitive to ligand binding (Kim et al., 1993).)

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(2), 5 mM NH(4)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.

Data Analysis

Parameters were obtained from steady-state kinetic or fluorescence studies by fitting the substrate saturation data to either the Michaelis-Menten equation for hyperbolic kinetics or the Hill equation for sigmoidal kinetics. Kinetic data for MgATP saturation at low Fru-6P concentration (Fig. 3B) were fit to the initial velocity equation for a sequential random kinetic mechanism assuming non-rapidequilibrium conditions (Segel, 1975; Ferdinand, 1966). The GDP and AMPPNP inhibition data were analyzed as follows: first, the inhibition pattern was identified (competitive, noncompetitive, etc.). Based on this identification, the inhibition data were fit, using nonlinear regression analysis, to the equation for either competitive or noncompetitive inhibition (Cleland, 1979). The parameters generated were used to construct linear equations for the double-reciprocal plots. Finally, slopes and/or intercepts of the inhibition lines were plotted against inhibitor concentration to determine the values for K and/or K. All inhibition data were corrected for loss of activity due to enzyme instability. Curve fitting was performed using the program INPLOT (GraphPad, Inc., San Diego, CA).


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(max)) versus MgATP concentration for native E. coli PFK fully activated with 2 mM GDP (bullet) and chimeric PFK (circle) (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(2) concentration was 20 mM.




RESULTS

The Chimeric Subunit

X-ray crystallography has revealed that the amino acid residues that bind Fru-6P are the same between the active sites of BsPFK and EcPFK (Evans et al., 1981; Shirakihara and Evans, 1988). Three of the residues that bind ATP are different between the two active sites. They are, for BsPFK versus EcPFK: Cys instead of Phe, Lys instead of Arg, and Gln instead of Met. In the chimeric subunit (Fig. 1), the BsPFK/EcPFK junction is at residue 122. The residues that bind ATP are from the BsPFK portion of the subunit, while those that bind Fru-6P are from the EcPFK portion. The region of the ATP-binding domain of EcPFK that moves (residues 71-95 and 101-118) during the open-to-closed transition (Shirakihara and Evans, 1988) has been replaced with the corresponding rigid region of BsPFK (Schirmer and Evans, 1990).


Figure 1: Schematic diagram of the chimeric PFK subunit. NH(2) 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.



Structural Properties

It is important to know something about the structural properties of the chimeric enzyme before investigating its kinetic and allosteric properties. SDS-polyacrylamide gel electrophoresis analysis indicates that the subunit molecular weights of the three enzymes are similar, being 36,000 ± 1,000 daltons. Results of a sucrose gradient sedimentation study show that ChiPFK exists as a tetramer 137,000 daltons in size. Sedimentation experiments performed under the same conditions for BsPFK and EcPFK give similar molecular weights of 139,000 and 136,000, respectively. Thus, the three enzymes exist as tetramers similar in size. Analysis of the ChiPFK sucrose gradient fractions by SDS-polyacrylamide gel electrophoresis followed by silver staining reveals that no dimers or monomers were present within the gradient. About 75% of the total enzyme activity loaded was recovered from the fractions collected in the sedimentation study.

Stability

Both activity and fluorescence measurements gave evidence that ChiPFK is somewhat unstable. Once diluted to a concentration appropriate for activity assays, ChiPFK began to lose activity over time: about 15% was lost over a period of 2 h. The activity of the diluted enzyme was unstable under a variety of buffer and temperature conditions. Instability was also observed in the fluorescence experiments in which Trp fluorescence was titrated by incremental addition of ligand solution. A saturable quenching of fluorescence was observed in these experiments regardless of whether ligand or simply buffer was added, suggesting that the effect is due to time-dependent inactivation, not ligand binding.

Kinetic Parameters

The chimeric enzyme was studied using steady-state kinetics, and the kinetic parameters obtained were compared to those for the two native enzymes. The results (Table 1) indicate that the chimeric enzyme is 2-fold less catalytically active than either of the native enzymes. However, its affinity for ATP as measured by K is similar. The larger value of K for EcPFK (103 µM) is most likely the result of substrate antagonism between MgATP and Fru-6P in the active site (Deville-Bonne et al., 1991b) since, when Fru-6P concentration is 50 µM instead of 1.5 mM, K drops 1.8-fold to 70 µM. (A similar drop in K occurs for ChiPFK when Fru-6P concentration is lowered from 0.3 mM to 0.05 mM, indicating that substrate antagonism is also present in ChiPFK.) The Fru-6P saturation curves in the presence of saturating MgATP concentration vary in degree of sigmoidicity among the three enzymes. Whereas Fru-6P saturation of EcPFK is highly sigmoidal (n is 5.8), it is hyperbolic for BsPFK (n is 1.1), and somewhat sigmoidal for ChiPFK (n is 1.7). The S value for ChiPFK is closer to the value for BsPFK than that for EcPFK. However, when EcPFK is activated by GDP (2 mM), the Fru-6P saturation curve becomes hyperbolic and Sfalls to 50 µM, which is close to the value for either BsPFK or ChiPFK. These results show that, in terms of its kinetic parameters, ChiPFK is similar to BsPFK and the activated form of EcPFK.



Fru-6P Saturation Kinetics

Fig. 2displays more clearly the differences among the three enzymes in terms of their saturation by Fru-6P in the presence of 1.0 mM MgATP. Whereas saturation of EcPFK is highly sigmoidal, saturation of ChiPFK is nearly hyperbolic. Thus, cooperativity has been largely destroyed in ChiPFK. Some remains, however (n(H) is 1.7; Fig. 2, inset). The residual cooperativity persists (n(H) is 1.8) even for Fru-6P saturation of ChiPFK in the presence of less-than-saturating MgATP concentration (50 µM; not shown). It is also present (n(H) is 1.5) when other nucleoside triphosphates (NTPs) such as GTP or CTP serve as the phosphate donor. Together, these results suggest that ChiPFK cooperative behavior is independent of the absolute amount of ATP present and the identity of the NTP. What appears to be important is the amount of MgATP (or other NTP) relative to the amount of Fru-6P present. When MgATP concentration was low (50 µM), ChiPFK activity did not increase with Fru-6P concentration beyond 50 µM. This result indicates that MgATP binding and/or release is not a rapid-equilibrium process for ChiPFK. Similar results showing non-rapid-equilibrium binding and/or release of MgATP were obtained for BsPFK and the activated form of EcPFK.


Figure 2: Dependence of PFK activity on Fru-6P concentration. circle, native B. stearothermophilus PFK; bullet, 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.



MgATP Saturation in the Presence of Low Fru-6P Concentration

MgATP saturation curves for ChiPFK and the two native enzymes obtained in the presence of saturating Fru-6P concentration are hyperbolic and yield similar K values (Table 1). However, when Fru-6P concentration is low (50 µM), differences become apparent between the MgATP saturation curves of EcPFK and ChiPFK (Fig. 3, A and B). Under this condition, significant substrate inhibition of EcPFK by MgATP is evident (Fig. 3A; Kundrot and Evans, 1991; Johnson and Reinhart, 1992). The left-most portion of the curve in Fig. 3A can be fit to the Michaelis-Menten equation (inset), yielding a K of 0.5 ± 0.1 µM. MgATP also inhibits ChiPFK at low Fru-6P concentration (Fig. 3B, open circles), but the inhibition is much less severe (K is 50 µM). Whereas MgATP concentrations above 1 millimolar begin to inhibit ChiPFK (open circles, Fig. 3B), concentrations of only 5 µmol inhibit unactivated-EcPFK (Fig. 3A). Thus, ChiPFK is 200-fold less sensitive to ATP inhibition than is EcPFK. Furthermore, the data for ChiPFK can be fit to the equation for a steady-state random Bi Bi kinetic mechanism (Segel, 1975) but those for unactivated-EcPFK cannot. The results suggest that the mechanisms of ATP inhibition of ChiPFK and EcPFK are different. However, whereas MgATP saturation curves at low Fru-6P concentration for ChiPFK and unactivated-EcPFK are dramatically different, those for ChiPFK (Fig. 3B, open circles) and EcPFK activated by 2 mM GDP (Fig. 3B, closed circles) are similar. These results suggest that the ChiPFK subunit is locked in a conformation that resembles the activated EcPFK subunit.

Heterotropic Regulation

The abilities of PEP to allosterically inhibit and GDP to allosterically activate ChiPFK were investigated using steady-state kinetics. The results obtained indicate that ChiPFK activity is insensitive to regulation by either effector. Fig. 4A shows that both native enzymes are inhibited by PEP, although the sensitivity and cooperativity of their responses differ. The inhibition profile for EcPFK is highly sigmoidal with a Hill number of 4.3 (I is 1.17 ± 0.01 mM), while the profile for BsPFK is much less sigmoidal with a Hill number of 1.6 (I is 0.28 ± 0.01 mM). However, PEP has essentially no effect on the chimeric enzyme. ChiPFK likewise cannot be activated by GDP. As shown in Fig. 4B, GDP strongly activates EcPFK when MgATP concentration is saturating and the Fru-6P concentration is equal to the S value (0.45 mM). The K for the activation, which is hyperbolic, is 13 ± 0.4 µM. On the other hand, GDP can either activate or inhibit BsPFK, depending on the concentration of GDP. In the presence of Fru-6P at a concentration equal to the S value (30 µM), as GDP concentration increases, it first activates BsPFK weakly to about 20% (K is 90 ± 10 µM), then begins to inhibit (Fig. 4B). Since the inhibition by GDP is competitive with respect to MgATP (K = 1.5 ± 0.2 mM; not shown), it is due to binding in the active site. (^3)The chimeric enzyme cannot be activated by GDP but, like BsPFK, is inhibited by it. However, the inhibition pattern is mixed-type noncompetitive with respect to MgATP (K = 4.3 ± 0.9 mM; K = 26 ± 6 mM) rather than competitive. The origin of the inhibition is not clear.


Figure 4: Effect of PEP and GDP on PFK activity. circle, B. stearothermophilus PFK; bullet, 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.



Thermal Inactivation Studies

A possible explanation for the lack of heterotropic regulation of ChiPFK by PEP and GDP is that these ligands cannot bind the effector site of the enzyme. To address this possibility, thermal inactivation experiments were performed in which ChiPFK was heated in the absence or presence of ligand. The results show that both Fru-6P and MgGDP protect native EcPFK against thermal inactivation at 60 °C (Fig. 5A). PEP also protects EcPFK, but the protection is partial, with 68% of activity remaining after 1 h. Similar experiments were performed on ChiPFK. (50 °C was used instead of 60 °C because of the lower stability of ChiPFK.) As shown in Fig. 5B, Fru-6P at 5 mM protects ChiPFK against thermal inactivation. This result contrasts with the inability of a 10-fold lower concentration of Fru-6P to stabilize ChiPFK diluted for activity assays (above). It suggests that ChiPFK dissociates along its ``active'' interface, and that Fru-6P prevents dissociation by interacting with Arg and Arg across it (Teschner et al., 1990). PEP also protects ChiPFK, to about the same extent it protects native EcPFK (64 versus 68% for EcPFK). Thus, PEP binds as well to ChiPFK as it does to EcPFK. In contrast, MgGDP offers little protection. Only 15% of activity remains after a 1-h incubation, compared to 9% in the absence of ligand (Fig. 5B). The simplest interpretation of this result is that MgGDP binds poorly in the effector site of ChiPFK.


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(2) and 2 mM DTT (at 60 and 50 °C for EcPFK and ChiPFK, respectively) in the presence of no ligand (bullet), 5 mM Fru-6P (circle), 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.



AMPPNP Inhibition Studies

Inhibition of both activated-EcPFK and ChiPFK by AMPPNP with respect to MgATP was competitive (K= 24 ± 2 and 20 ± 2 µM, respectively), as was AMPPNP inhibition of BsPFK with respect to MgATP (K of 50 ± 20 µM; Byrnes et al, 1994). Thus, as expected, AMPPNP competes with MgATP for binding in the active site. Interestingly, AMPPNP inhibition of both activated-EcPFK and ChiPFK with respect to Fru-6P gave similar competitive-like patterns of lines that intersected just to the right of the 1/velocity axis in double-reciprocal plots. (K values of 7 ± 7 and 9 ± 6 µM were obtained for activated-EcPFK and ChiPFK, respectively.) In contrast, AMPPNP inhibition of BsPFK with respect to Fru-6P is noncompetitive (K = 0.29 ± 0.09 mM and K = 0.40 ± 0.12 mM; Byrnes et al., 1994). Thus, AMPPNP binding to activated EcPFK and ChiPFK is different from its binding to BsPFK. The competitive-like inhibition with respect to Fru-6P could be due to antagonism of Fru-6P binding by AMPPNP in the active site (ATP shows a similar effect that is less pronounced). In addition, as shown in Fig. 6, the Hill number for the Fru-6P saturation curve obtained in the presence of AMPPNP decreases with increasing AMPPNP concentration until it reaches 1.0 at 200 µM AMPPNP. Thus, AMPPNP abolishes the sigmoidicity of the Fru-6P saturation curve of ChiPFK.


Figure 6: Effect of AMPPNP on the Fru-6P-dependent cooperative behavior of chimeric PFK. Plot of Hill number (bullet) or S value (circle) 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.



Effect of Ligand Binding on TrpIntrinsic Fluorescence

Two types of steady-state fluorescence experiments were performed. In the first type, the intrinsic fluorescence of EcPFK was titrated by addition of small volumes of ligand to the enzyme solution. (These experiments could not be done on ChiPFK because of its instability over time.) The results are presented in Table 2. Titration of EcPFK fluorescence with Fru-6P, with AMPPNP, and with AMPPNP after incubation in the presence of Fru-6P (Table 2) gave results similar to those previously reported (Deville-Bonne and Garel, 1992; Johnson and Reinhart, 1992). In the second type of experiment, a saturating amount of ligand was added to the enzyme solution, and the change in fluorescence intensity measured (Table 3). The results show that AMPPNP (or ATP) binding cause the same fluorescence increase (12%) in both ChiPFK and EcPFK, but the fluorescence decrease induced in ChiPFK upon Fru-6P binding (8%) was less than that induced in EcPFK (19%). ChiPFK can thus exist in two conformational states: a high fluorescence state and a low fluorescence state. Addition of Fru-6P to EcPFK incubated in the presence of AMPPNP at concentrations as low as 5 µM resulted in no fluorescence change (Table 3; AMPPNP concentrations below 2 µM did allow Fru-6P (500 µM) to bind, however). This dramatic blockage of Fru-6P binding by AMPPNP, which may indicate closure of the EcPFK active site upon AMPPNP binding, was not seen in ChiPFK (Table 3). Finally, in order to investigate the combined effects of Fru-6P and AMPPNP on the fluorescence of ChiPFK, experiments were performed in which a saturating amount of one ligand was added to the enzyme in the presence of increasing amounts of the other. The results indicate that a saturating amount of Fru-6P (500 µM) cannot bring ChiPFK completely to the low fluorescence state when AMPPNP is present, even at low levels (1 µM). This inability of Fru-6P to bring ChiPFK completely to its low fluorescence state was more evident at higher AMPPNP levels. On the other hand, AMPPNP was found to be more effective than Fru-6P in its ability to change the conformational state of ChiPFK. At Fru-6P levels as high as 100 µM, a saturating amount of AMPPNP could bring ChiPFK fully to its high fluorescence state. Only at very high levels of Fru-6P (>500 µM) was this effect by AMPPNP not observed.






DISCUSSION

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.^3 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 (ATPS) 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).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 31676. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to the memory of David W. H. Chang(1971-1991).

§
Present address: Dept. of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. This paper was adapted from a dissertation written in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Supported in part by a grant to Louisiana State University from the Howard Hughes Medical Institute.

**
To whom correspondence should be addressed: Dept. of Biochemistry, Rm. 322 Choppin Hall, Louisiana State University, Baton Rouge, LA 70803. Fax: 504-388-5321.

(^1)
The abbreviations used are: PFK, phosphofructokinase (Fru-6P 1-phosphotransferase); BsPFK and EcPFK, the phosphofructokinases from Bacillus stearothermophilus and Escherichia coli, respectively; ChiPFK, the chimeric phosphofructokinase; Fru-6P, fructose 6-phosphate; PEP, phosphoenolpyruvate; AMPPNP, adenylyl imidodiphosphate; DTT, dithiothreitol; ATPS, 5`-[-thio]triphosphate.

(^2)
It was reasoned that this mutation would not significantly alter the properties of the chimeric enzyme since 1) the amino acid change is not a drastic one, and 2) there is already a degree of variation between the native enzymes at this position (valine versus leucine).

(^3)
Zhu, X., Byrnes, M., Nelson, J. W., and Chang, S. H. Biochemistry, in press.


ACKNOWLEDGEMENTS

We thank Drs. Isiah Warner and Mary Barkley for the use of their fluorescence spectrometers, and Dr. Noni H. Byrnes for help with the fluorescence studies. We are also grateful to Dr. Ben Valdez for reading the manuscript.


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