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INTRODUCTION |
The bifunctional enzyme
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
(6PF-2K/Fru-2,6-P2ase)1
is a homodimer, and each subunit contains an N-terminal kinase domain
and a C-terminal bisphosphatase domain (1, 2). The C-terminal
Fru-2,6-P2ase domain is homologous to the glycerate mutase
and acid phosphatase families (3-5). Fru-2,6-P2ase
catalyzes the hydrolysis of Fru-2,6-P2 by the formation of
a phosphoenzyme intermediate through phosphorylated His-258.
Residue His-258 along with Glu-327 and His-392 comprise the catalytic
triad in rat liver Fru-2,6-P2ase (reviewed in Ref 2).
Recent work using x-ray crystallography and NMR supports the importance
of these residues and provides further insights into the reaction
mechanism (4, 6, 7). In addition, the structural study by Hasemann
et al. (5) and the modeling work by Bertrand et
al. (8) have demonstrated independently that the N-terminal 6PF-2K
domain is structurally related to mononucleotide-binding proteins, such as adenylate kinase and the catalytic cores of G proteins. Based on the
structural comparison, Hasemann et al. (5) further proposed that the 6PF-2K domain, like G proteins, operates via a transition state stabilization mechanism and predicted additional residues important for substrate binding or catalysis, which were subsequently confirmed by mutagenesis (9, 10).
It has long been recognized that both the N and C termini of the enzyme
play important roles in the regulation of the 6PF-2K and the
Fru-2,6-P2ase activities (11). Recently, Wu et
al. (12) found that the charged residues as well as
Ser20-Ser24 in the N-terminal tail might be
involved in interactions with the catalytic domains. The interaction
may lead to increased 6PF-2K and reduced Fru-2,6-P2ase
activities, as disruption of this N-terminal interaction resulted in
the reduction in both kinase and phosphatase activities. The work of
Kurland et al. (13) revealed that residues Gly5-Glu6-Leu7 of the liver isoform
are responsible for the increase in the affinity of 6PF-2K for Fru-6-P,
the inhibition of Fru-2,6-P2ase activity, and the effects
of cAMP-dependent protein kinase phosphorylation on
the two activities. The C terminus of the bifunctional enzyme has a
regulatory function on both the kinase and bisphosphatase activities,
and it is generally regarded as having an inhibitory effect on the
bisphosphatase activity (14-17).
The kinase domain of the bifunctional enzyme has a Walker A motif
(Gly-X-X-X-X-Gly-Lys-Thr)
and B motif (Z-Z-Z-Z-Asp), which are typical of
nucleotide-binding proteins (3, 18, 19). Mutation of the first Gly of
the A motif abolished the kinase activity of the rat liver bifunctional
enzyme (20). The Lys residue in the A motif is critical for ATP
binding, and the Thr residue is necessary for catalysis (21, 10). The
Asp residue in the B motif is also important for the binding of ATP, as
it coordinates the ATP-bound Mg2+ (18). Interestingly, a
sequence characteristic of the Walker A motif was also identified in
the Fru-2,6-P2ase domain (22). Early work on rat hepatic
enzyme revealed that the isolated Fru-2,6-P2ase domain can
be regulated by GTP, ATP, and other nucleotide triphosphates, and
Arg-360 was important for the regulation of Fru-2,6-P2ase activity by nucleotide triphosphates (22). These results suggest that
the nucleotide triphosphates might regulate the activity of
Fru-2,6-P2ase by binding specifically to this domain. In
addition, it has been found that 6PF-2K of chicken hepatic enzyme was
activated by substrate ATP. The Hill plot of the kinase activity
against the concentrations of ATP yielded a coefficient of 0.56 (23). Such a phenomenon might be either the result of negative cooperativity between the two identical ATP binding sites in the kinase domains of
the homodimeric enzyme or the allosteric binding of ATP to the
Fru-2,6-P2ase domain. In this work, we provide evidence to support the latter possibility by using site-directed mutagenesis and
biochemical approaches.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction endonucleases and other DNA modifying
enzymes were obtained from Life Technologies, Inc. or New England
Biolabs. ATP, Fru-6-P, and Fru-2,6-P2 were purchased from
Sigma. N-methylisatoic anhydride was purchased from Aldrich.
Construction of Expression Plasmids and Protein
Preparation--
Point mutations were made using standard polymerase
chain reaction techniques and checked by double strand DNA sequencing. The construct for chimeric enzyme, where the last 25 residues from the
C terminus of CKB was replaced with that of RKB (CKB-RCT), was
generated as reported elsewhere (17). All forms of CKB were expressed in Escherichia coli BL21(DE3) using
pET3a plasmid and purified to homogeneity by the same
procedures for the preparation of the wild type CKB (24, 25).
Preparation of Mant-ATP--
The N-methylanthraniloyl
derivative of ATP was synthesized according to Hiratsuda (26). After
reaction, the product was purified according to Woodward et
al. (27), except that a DEAE-Sephadex A-25 column was used instead
of a DEAE-cellulose column. The purified Mant-ATP was verified by
absorption spectra, fluorescence spectra, and thin-layer chromatography
(data not shown).
Determination of Circular Dichroism Spectra--
All far UV
spectra were collected on a Jasco 720 dichrograph in a 0.01-cm cell at
25 °C. Scans were collected at 50 nm/min between 190 and 250 nm with
a response of 2 s and resolution of 0.2 nm. The bandwidth was 1 nm. All data were the average of four blank corrected samples. The
proteins were equilibrated with buffer containing 20 mM
Tris-HCl, pH 8.0, 0.5 mM EDTA, 50 mM KCl, and 1 mM DTT by mini-gel filtration. Samples were then
centrifuged for 10 min at 15,000 × g to remove any
precipitates, and the A280 was measured to scale
the CD data to the same concentration.
Fluorescence Measurements--
Different forms of CKB (final
concentration, 20 µg/ml) were incubated for 20 min at 25 °C with
various concentrations of GdnHCl in 50 mM Tris-HCl, pH 7.5, and 1 mM DTT. Protein fluorescence was measured with a
Hitachi F-4010 fluorescence spectrophotometer from 300 to 400 nm with
an excitation wavelength of 295 nm. The emission fluorescence intensity
at 335 nm and the maximum emission wavelength were recorded.
Fluorescence Determination of Ligand Binding--
The increase
in the extrinsic fluorescence was employed to measure the binding of
Mant-ATP to different enzyme forms at 20 °C with an Hitachi F-4010
fluorescence spectrophotometer. The spectral bandwidths were 3 and 5 nm, respectively, for excitation and emission. Mant-ATP (50 µM-5 mM) was added dropwise (1-2 µl) to
90-100 µg of enzyme diluted in 1 ml of buffer containing 100 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM Pi, 2 mM DTT, and 5 mM MgCl2. The sample was excited at 350 nm, and
the fluorescence was scanned from 400 to 500 nm. The fluorescence of
free Mant-ATP in the absence of enzyme was also recorded under the same
conditions. The differential spectrum resulted from subtracting the
fluorescence of Mant-ATP in the absence of enzyme from that in the
presence enzyme was integrated. All measurements were corrected for
dilution (less than 3%) and inner filter effects.
6PF-2K Activity Determination--
6PF-2K activity was assayed
by the formation of Fru-2,6-P2, which was quantified by the
stimulation of potato tuber pyrophosphate (fructose-6-phosphate
phosphotransferase) (28). Unless stated otherwise, the reaction mixture
contained 100 mM Tris-HCl, pH 7.4, 5 mM
Pi, 1 mM DTT, 10 mM ATP, 10 mM MgCl2, and 2 mM Fru-6-P in a
final volume of 50 µl. The reaction was initiated by the addition of
enzyme. The mixture was incubated for 10 min at 30 °C and terminated
with the addition of 1.5 M NaOH. The solution was heated
for 1 h at 80 °C and diluted to 1 ml with water. Suitable aliquots of the diluted solution were then assayed for
Fru-2,6-P2.
Fru-2,6-P2ase Activity Determination--
The
activity of Fru-2,6-P2ase was assayed at pH 7.4 by
following the rate of production of [32P]Pi
from [2-32P]Fru-2,6-P2 as described
previously by El-Maghrabi et al. (29).
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RESULTS |
Effects of Mutation of Arg-279 and Arg-359 of CKB on the Properties
of Fru-2,6-P2ase--
Our early work showed that the
6PF-2K activity of the chicken liver bifunctional enzyme was stimulated
by its substrate ATP, indicating an apparent negative cooperativity for
the enzyme in binding of ATP (23, 24). Because
6PF-2K/Fru-2,6-P2ase is a homodimer, such cooperativity
might result from the interaction between the two ATP binding sites of
the kinase domains of the CKB homodimer (23, 24). Alternatively, the
cooperativity may result from the interaction between the ATP binding
site of the kinase domain and that of the bisphosphatase domain. To
determine which mechanism underlies the ATP activation of chicken
6PF-2K, we examined whether elimination of the binding of ATP to the
bisphosphatase domain affected the activation of chicken 6PF-2K.
Previously, it was reported that the Fru-2,6-P2ase
activities of the hepatic bifunctional enzyme was regulated by
triphosphate nucleotides such as GTP or ATP (22, 28). Lee et
al. (22) reported that the activation of the
Fru-2,6-P2ase by GTP or ATP involved a direct interaction
of the triphosphate nucleotides with the active site of the
bisphosphatase domain, and the activation is caused by the phosphate
moieties of the triphosphate nucleotides competing with the
2-phospho group of Fru-2,6-P2 for the
phosphoenzyme interaction, thus relieving substrate inhibition.
Arg-360 of rat liver enzyme was demonstrated as a critical
residue responsible for the substrate inhibition (30) and is important
for the binding of GTP to the bisphosphatase domain (22). As shown in
Table I, this residue is highly conserved
among various isoforms of the bifunctional enzymes; the corresponding
residue of the chicken liver enzyme is Arg-359. In addition, the
nucleotide-binding motif (Gly274-Leu-Ser-Ala-Arg-Gly-Lys-Gln281) found
in the bisphosphatase domain of RKB (22) is also conserved in CKB
(Table I). The penultimate basic residue was shown previously to be
critical for nucleotide binding (31). The corresponding residue in the
chicken liver enzyme is Arg-279 (Table I). To investigate whether
Arg-279 and/or Arg-359 are involved in the binding of ATP to the
bisphosphatase domain of CKB, two mutants, CKBR279A and
CKBR359A, were produced and analyzed.
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Table I
Comparison of the Walker A motif and the sequence around Arg-359 in the
bisphosphatase domain of CKB with the corresponding sequences in other
isoforms of the bifunctional enzyme
The Walker A motif in the bisphosphatase domain of CKB is underlined,
and the two basic amino acid residues, Arg-279 and Arg-359, are in bold
face type. The corresponding residues in RKB are Lys-280 and Arg-360,
respectively.
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The mutant enzymes CKBR279A and CKBR359A were
expressed in E. coli and purified to homogeneity as
described previously (24, 25). We first examined the
Fru-2,6-P2ase activities of the two mutant enzymes. As shown in Table II,
CKBR359A has a Km value for
Fru-2,6-P2 that is 6.5-fold higher than the wild type CKB,
and substrate inhibition of CKBR359A by
Fru-2,6-P2 was also diminished (Fig.
1). These results are consistent with
those of the corresponding RKB mutant, RKBR360A (30),
suggesting that Arg-359, like Arg-360 in the rat enzyme, is a critical
residue in the chicken Fru-2,6-P2ase domain for triphosphate nucleotide binding. On the other hand,
CKBR279A showed Vmax and
Km values for Fru-2,6-P2 similar to those of the wild type CKB and acted similarly to the wild type enzyme
with regard to substrate inhibition (Fig. 1). However, the
Fru-2,6-P2ase activity of CKBR279A, unlike the
wild type enzyme, could not be activated by ATP (Fig. 2), suggesting that Arg-279 of CKB is
required for ATP binding to the Fru-2,6-P2ase domain.
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Table II
Comparison of kinetic parameters of the wild type and mutant forms of
CKB and RKB
The 6PF-2K activity was measured in buffer containing 100 mM Tris-HCl, pH 7.4, 5 mM Pi, 1 mM DTT, and 10 mM MgCl2.
Km values for ATP or Fru-6-P were determined in the
presence of 2 mM Fru-6-P or 10 mM ATP,
respectively. Vmax and Km values
were obtained using a hyperbolic regression analysis program. For the
wild type CKB, fitting the data corresponding to the two ATP ranges
(0.05-0.4 and 0.4-4 mM) separately into the above
mentioned software yielded two Vmax and,
correspondingly, two Km values. The values represent
the mean ± S.D. for three determinations.
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Fig. 1.
Fru-2,6-P2-dose dependence of the
Fru-2,6-P2ase activity of the mutant and wild type
enzymes. Fru-2,6-P2ase activity was assayed by
determining the rates of production of 32P from
[2-32P]Fru-2,6-P2 as described under
"Experimental Procedures." The concentrations of
[2-32P]Fru-2,6-P2 are 0.1 µM
(white bars), 5 µM (gray bars), and
20 µM (black bars), respectively. Full
Fru-2,6-P2ase activities (100%) are 14, 15, and 14 milliunits/mg for the wild type CKB, CKBR279A, and
CKBR359A, respectively. Assays were performed in duplicate,
and the results represent two separate experiments.
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Fig. 2.
Effects of ATP and Pi on the
Fru-2,6- P2ase activity of CKB and
the wild type CKB. Fru-2,6-P2ase activity was assayed
using 20 µM Fru-2,6-P2 in the presence or
absence of ATP or Pi. Assays were performed in duplicate,
and the results represent two separate experiments.
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Effects of Mutation of Arg-279 and Arg-359 on ATP Activation of
Chicken Liver 6PF-2K--
Knowing that the two Arg residues are
important for the Fru-2,6-P2ase domain to bind ATP,
an important question is whether the two mutations affect the
activation of the 6PF-2K of CKB by ATP. The 6PF-2K of the wild type CKB
exhibited substrate activation by ATP, as reported earlier when assayed
in the presence of 1-2 mM of MgCl2 (23, 24).
Although MgCl2 at concentrations over 2 mM
inhibits the chicken liver 6PF-2K, it does not affect the substrate
activation of 6PF-2K of wild type CKB by ATP, as the ATP activation was
also observed in the presence of 10 mM MgCl2 (Fig. 3). For convenience, all of the
following kinetic analyses were carried out in buffers containing 10 mM MgCl2. The double reciprocal plot of 6PF-2K
activity of CKB versus the ATP concentrations revealed two
slopes (Fig. 3), which yielded two pairs of Km and
Vmax values corresponding to the nonactivated
and the ATP-activated activities of the 6PF-2K of CKB, respectively
(Table II). In contrast, neither CKBR279A nor
CKBR359A exhibited ATP activation (Fig. 3). Because these
two mutations did not affect the Km of the 6PF-2K
for Fru-6-P (Table II), it is likely that the ATP activation of the
chicken liver 6PF-2K is caused by allosteric binding of ATP to the
Fru-2,6-P2ase domain.

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Fig. 3.
Lineweaver-Burk plots of the 6PF-2K
activities of CKB, CKBR359A, and the wild type
CKB versus ATP concentrations. The 6PF-2K
activities of three enzymes were assayed at 30 °C in a buffer
containing 100 mM Tris-HCl, pH 7.4, 5 mM
Pi, 1 mM DTT, 2 mM Fru-6-P, and 10 mM MgCl2. The results are representative of at
least four experiments.
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CD Spectra and Fluorescence Spectra of CKB279A and
CKB359A--
To determine whether any of these two
mutations grossly alters the secondary structure of the enzyme, the
circular dichroic spectra of the mutant and wild type forms of CKB were
examined. As shown in Fig. 4, the
circular dichroic spectra of various forms of CKB were very similar:
The wild type CKB may contain 29.1 ± 1.7%
-helix, 27.3 ± 1.3%
-structure, and 10.2 ± 1.2% turn;
CKBR279A may contain 30.4 ± 1.7%
-helix,
26.9 ± 1.3%
-structure, and 10.7 ± 1.2% turn; and
CKBR359A may contain 28.7 ± 1.6%
-helix,
27.6 ± 1.5%
-structure and 11.3 ± 0.9% turn. These
data indicated that the mutations are unlikely to cause significant
changes in the secondary structure of the enzyme. This conclusion was
further confirmed by the fluorescence spectroscopic analysis. Fig.
5 shows that there were no significant differences between either of the mutant forms and the wild type CKB in
fluorescence quenching or red shift of the emission maximum caused by
GdnHCl.

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Fig. 4.
Circular dichroism spectra of mutant and wild
type CKB. The circular dichroism data were blank-corrected and
scaled based on the protein concentration. The Mol degree
(mdeg) of signal corresponds to the measurements conducted
in an 0.01-cm cell with a protein solution equal to 0.8 A280 in a 1-cm light path. Only selected data
points are presented for the sake of clarity.
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Fig. 5.
GdnHCl-induced changes in fluorescence of the
mutant and wild type CKB. Each protein (final concentration, 20 µg/ml) was incubated for 20 min at 25 °C with various
concentrations of GdnHCl. The fluorescence was measured with the
excitation wavelength of 295 nm. The emission maximum (upper
panel) and fluorescence intensity at 335 nm (lower
panel) for CKB ( ), CKBR279A ( ),
CKBR359A ( ), and CKB-RCT ( ) are shown. Assays were
performed in duplicate, and the results represent at least two separate
experiments.
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Binding Analysis Employing Mant-ATP--
Although the analysis of
the kinetic parameters of the two mutants suggested that the binding of
ATP to the Fru-2,6-P2ase domain regulates the 6PF-2K
activity allosterically, direct measurement of ATP binding of the
enzyme should provide more straightforward evidence. Thus the ATP
binding properties of these different forms of CKB were investigated by
fluorescence spectroscopy, using Mant-ATP as ligand.
Mant-ATP had been previously used to study the ATP binding properties
of RKB and the rat testis enzyme (21, 32). CKB could utilize Mant-ATP
as phosphate donor with a Km value of 14 µM and Vmax of 75 milliunits/mg.
In addition, the 6PF-2K activities were inhibited by Mant-ATP at higher
concentrations (>0.1 mM) (data not shown), consistent with
the report on the rat testis enzyme (32).
The addition of CKB resulted in an increase of the fluorescence of the
Mant-ATP under the conditions described under "Experimental Procedures." Such an increase caused by enzyme was used to analyze the binding of enzymes to Mant-ATP. A Scatchard plot of
F
(the fluorescence change) versus
F/(Mant-ATP) (the
fluorescence change over the concentration of Mant-ATP) showed that the
wild type CKB exhibits an apparent negative cooperativity for binding
Mant-ATP. As shown in Fig. 6, it seems
that the wild type enzyme has two kinds of sites with different
affinities for binding of ATP, as there were the two distinct slopes
for ATP binding showing two different dissociation constants
(Kd) (Table III).
However, CKBR279A and CKBR359A did not show
cooperativity in binding Mant-ATP (Fig. 6), and there is only one
Kd value of CKBR279A or
CKBR359A, which is similar to the high affinity
value of the wild type CKB (Table III). We also tested the
binding of the isolated bisphosphatase domain of CKB (CBD) with
Mant-ATP. As expected, CBD bound Mant-ATP with one
Kd value equivalent to the low affinity value of CKB (Fig. 6, Table III). These data indicate that the mutation of
Arg-359 or Arg-279 to Ala eliminates both the binding of ATP to the
bisphosphatase domain of the bifunctional enzyme and the activation of
6PF-2K by ATP in CKB and that the Walker A motif in the bisphosphatase
domain is critical for the binding of ATP. Considering these together
with the kinetic data, it is reasonable to conclude that the ATP
activation of the chicken liver 6PF-2K is caused by the allosteric
binding of ATP to the Fru-2,6-P2ase domain of the
enzyme.

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Fig. 6.
Scatchard plots of Mant-ATP binding. The
increase in the extrinsic fluorescence was used to measure the binding
of Mant-ATP to different enzymes as described under "Experimental
Procedures." The differential spectrum resulted from subtracting the
fluorescence spectrum of Mant-ATP in the absence of enzyme from that in
the presence of enzyme was integrated as F.
The results are representative of at least three experiments.
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Table III
Dissociation constants of Mant-ATP to the wild type and mutant enzymes
The binding was measured by fluorescence spectroscopy as indicated
under "Experimental Procedures." The dissociation constants
(Kd) were calculated from the double reciprocal plot
of the values of integrated differential fluorescence intensity against
the corresponding concentrations of Mant-ATP using a hyperbolic
regression analysis program. The data were the means of three
determinations.
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Effect of the Replacement of C-terminal Tail of CKB with That of
RKB in the 6PF-2K Properties of the Enzyme--
Despite the high
homology between RKB and CKB, ATP activation of 6PF-2K was not observed
in RKB. It raised the question of whether this difference is due to the
divergence in their amino acid sequences at the C and N termini, in
which the majority of amino acid sequence differences between the two
enzymes are found. Recently, we found that a chimeric enzyme, CKB-RCT,
in which the C-terminal sequence of CKB was replaced with that of the
RKB, mimicked RKB in all of the kinetic properties of the
Fru-2,6-P2ase activity (17). It was interesting to see
whether the replacement of the C-terminal tail also affects the kinetic
behavior of the N-terminal 6PF-2K. In comparison with the wild type CKB
and RKB, CKB-RCT was more like RKB than CKB in the 6PF-2K properties;
particularly, the 6PF-2K of CKB-RCT could not be activated by substrate
ATP (Table II and Fig.
7A).

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Fig. 7.
Comparison of kinetic and fluorescence
properties of CKB, RKB, and CKB-RCT in ATP binding. A,
double reciprocal plots of the 6PF-2K activities of various enzymes
versus ATP concentrations; B, Scatchard plots of
fluorescence intensity increases of Mant-ATP caused by enzyme binding.
See Fig. 6 legend for description. The results are
representative of at least three experiments.
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Regardless of their different kinetic properties in the 6PF-2K
activity, CKB, RKB, and CKB-RCT had similar properties in binding to
Mant-ATP, as shown in Fig. 7B and Table III. Thus, the
difference in the C-terminal tails between these two hepatic enzymes
may be accounted for by the differences in the kinetic properties of
the 6PF-2K in respect to ATP. The two Kd values of RKB for Mant-ATP were in the same ranges as those of CKB (Table III),
and the Kd value for the high affinity binding was also similar to that of the rat testis enzyme (32). However, these
values were different from those of RKB as reported by Vertommen et al. (21), probably because of different assay conditions.
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DISCUSSION |
Two models, the pre-existent asymmetric model and the
ligand-induced sequential model, have been proposed to explain the
apparent negative cooperativity (33). The present study supports the pre-existent asymmetric model for the binding of ATP to the
bifunctional enzymes. It is the allosteric binding of ATP to the
bisphosphatase domain that causes the activation of 6PF-2K in CKB. The
binding of the bisphosphatase domain to ATP is specific because it has a Walker A motif, which is typical of nucleotide-binding. In addition, Arg-279, a basic residue within this motif, has here been proved vital
for binding of ATP. Another basic residue, Arg-359, is equally important for the binding of ATP to the bisphosphatase domain; this was
suggested initially by Lee et al. (22) and confirmed by our
present work. Although the Fru-2,6-P2ase domain
shares homology with proteins of the acid phosphatases and
phosphoglycerate mutase families, neither acid phosphatases and
phosphoglycerate mutases possess the nucleotide-binding motif (3). The
appearance of the nucleotide-binding sequence
(273Gly-Leu-Ser-Thr-Arg-Gly-Arg-Gln280 in CKB)
in the bisphosphatase domain of the bifunctional enzyme might be an
event that occurs later in evolution to meet the demand of the
multiple and subtle regulations of the critical bifunctional enzyme.
High concentrations of ATP favor the binding of ATP to the allosteric
binding site in the bisphosphatase domain, which might induce a global
structural change of that domain, and this in turn affects the
catalytic core of the kinase domain, resulting in the activation of
6PF-2K. CKB is highly homologous to all documented mammalian
6PF-2K/Fru-2,6-P2ases. For example, CKB and RKB share about
88% amino acid sequence identity (34). Although RKB also exhibited
negative cooperativity in ATP binding as revealed by a Mant-ATP binding
analysis (Fig. 7B), this isoform did not show ATP activation
of 6PF-2K (Fig. 7A). Interestingly, ATP activation of 6PF-2K
was not observed on the chimeric enzyme CKB-RCT, where the C-terminal
tail of CKB was replaced with that of RKB (Fig. 7). Therefore, it is
reasonable to believe that the C-terminal region of CKB is involved in
the activation of the 6PF-2K by the binding of ATP to the
bisphosphatase domain and that the differences in the C-terminal amino
acid sequences between the chicken and rat enzyme are responsible for
the differences in their kinetic properties in respect to ATP. In
addition, our recent work had revealed a role of the C-terminal tail of
CKB in the mediation of the repressive effect of the kinase domain on
the Fru-2,6-P2ase activity of the
enzyme.2 Taken together,
these data demonstrate the importance of the C-terminal tail in the
modulation of the 6PF-2K activity by the bisphosphatase domain, and
vice versa. Although the N-terminal tail of bifunctional enzyme is also
critically involved in the regulation of enzyme activities, its role in
the ATP-activation of chicken liver 6PF-2K remains to be determined.
The Vmax ratio of 6PF-2K to
Fru-2,6-P2ase was 6-13 for CKB, which was 2-4-fold higher
than that for RKB (Table II), indicating that CKB behaves more like a
6PF-2K comparing with RKB. In addition, the 6PF-2K of CKB is activated
by high concentrations of ATP whereas RKB cannot, and the inhibition of
6PF-2K activity of CKB by cAMP-dependent protein
kinase-catalyzed phosphorylation is much less than that of
RKB.2 Thus, the 6PF-2K activity is favored under
physiological conditions. This idea is consistent with the observation
that the chicken liver has a higher capacity for glucose utilization
than the rat liver (35, 36).