From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011
Received for publication, December 5, 2002
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ABSTRACT |
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Fructose-1,6-bisphosphatase requires divalent
cations (Mg2+, Mn2+, or Zn2+)
for catalysis, but a diverse set of monovalent cations (K+,
Tl+, Rb+, or
NH Fructose-1,6-bisphosphatase
(FBPase,1 EC 3.1.3.11)
hydrolyzes fructose 1,6-bisphosphate (F16P2) to fructose
6-phosphate (F6P) and phosphate (Pi) (1-6). Fructose
2,6-bisphosphate (F26P2) and AMP synergistically inhibit
FBPase. AMP inhibits by way of an allosteric and cooperative mechanism
with a Hill coefficient of 2 (7-9). F26P2 competes with
F16P2 for the active site (10-12). Coordinated regulation
of glycolysis and gluconeogenesis occurs in vivo, largely
because of opposite effects caused by F26P2 on FBPase
(inhibition) and fructose 6-phosphate 1-kinase (activation). Divalent
cations (Mg2+, Mn2+, and/or Zn2+)
are an absolute requirement for FBPase activity. Enzyme activity increases sigmoidally as a function of divalent cation concentration at
pH 7.5 (Hill coefficient of ~2), but at pH 9.6 the variation is
hyperbolic (8, 14).
The mammalian enzyme is a homotetramer and exists in distinct
conformational states, depending on the relative concentrations of
active site ligands and AMP (15). With or without metal cofactors and/or other active site ligands, but in the absence of AMP, FBPase is
in its R-state (16, 17). In the presence of AMP, however, the top pair
of subunits rotates 17° relative to the bottom pair, resulting in the
T-state conformer (18-20). The minimum distance separating AMP
molecules from any given active site is ~28 Å (18-20). Yet studies
in kinetics, NMR, fluorescence, and x-ray crystallography reveal
competition between AMP and divalent cations (11, 20-22).
Loop 52 In addition to the absolute requirement for divalent cations, certain
monovalent cations (K+ and Tl+ among others)
further enhance catalysis by FBPase (3, 5, 13). The precise mechanism
by which monovalent cations exert their influence, however, has yet to
be determined. Monovalent cation activation is in some fashion related
to loop 52 Previous work of Lipscomb and co-workers (27) focused on the
association of Tl+ and K+ with FBPase in the
absence of divalent cations and/or in the presence of AMP. Under these
conditions FBPase is inactive. Their investigation clearly shows the
association of Tl+ and K+ at metal loci 1, 2, and 3, usually the observed binding sites for essential divalent
activators. But how do monovalent cations interact with FBPase under
conditions that support catalysis? Presented below are a series of
product complexes of FBPase in the presence of Mg2+ and
Tl+. FBPase here is co-crystallized from an equilibrium
mixture of products and reactants, containing Mg2+ at a
saturating concentration and Tl+ ranging from zero to
70-fold in excess of its observed Ka value. Under
these conditions of crystallization FBPase is active. In the presence
of Mg2+, Tl+ no longer occupies metal sites 1 and 2, but interacts at four sites. Two of the four sites are mutually
exclusive with Mg2+ at site 3, whereas Tl+
interaction at two other sites could co-exist with Mg2+ at
sites 1 Materials--
F16P2, F26P2,
NADP+, and AMP were purchased from Sigma.
Glucose-6-phosphate dehydrogenase and phosphoglucose isomerase were from Roche Molecular Biochemicals. Other chemicals were of reagent grade or equivalent. The FBPase-deficient Escherichia coli
strain DF657 came from the Genetic Stock Center at Yale University.
Expression and Purification of FBPase--
Expression and
purification of FBPase followed the procedures of Burton et
al. (28) with minor modifications (20). E. coli strain
DF657, deficient in FBPase, was used in order to avoid contamination of
recombinant FBPase by endogenous enzyme. Protein purity and
concentration was confirmed by SDS-polyacrylamide gel electrophoresis
(29) and by the Bradford assay (30), respectively.
Crystallization of FBPase--
Crystals of FBPase were grown by
the method of hanging drops. Crystals of R-state complexes grew from
equal parts of a protein solution (10 mg/ml FBPase, 10 mM
KPi, pH 7.4, 5 mM MgCl2, 5 mM F6P, with or without 0.2 mM EDTA, in
different concentrations of thallium acetate (0, 1, 5, 20, or 100 mM)) and a precipitant solution (100 mM Hepes
pH 7.0, 5% t-butyl alcohol, 27% (v/v) glycerol, and 8%
(w/v) polyethylene glycol 3350). Crystals of T-state complexes grew
from 10 mg/ml FBPase, 10 mM KPi, pH 7.4, 5 mM MgCl2, 20 mM of thallium
acetate, 5 mM F6P, and 5 mM of AMP and a
precipitant solution (100 mM Hepes pH 7.0, 5%
t-butyl alcohol, and 10% (w/v) polyethylene glycol 3350).
The droplet volume was 4 µl. Wells contained 500 µl of the
precipitant solution. Crystals with dimensions of 0.2 × 0.2 × 0.2 mm grew in 3 days at room temperature. Conditions of
crystallization for R-state FBPase differ from those of previous studies (17, 20) and result in crystals that in some cases diffract to
near atomic resolution (see below).
Data Collection--
Data from FBPase complexes with 20 mM Tl+ (T-state and R-state) were collected at
synchrotron beam line X12C, Brookhaven National Laboratory, using a CCD
detector developed by Brandeis University, at a temperature of 100 K. The energy (12658 eV) of x-rays coincided with the atomic absorption
edge of Tl+. Data from the crystalline complex with 1 mM of Tl+ were collected at synchrotron beam
line X4A, Brookhaven National Laboratory on an ADSC, CCD detector at a
temperature of 100 K and an energy of 12658 eV. Data from the
crystalline complex with 100 mM Tl+ were
collected at synchrotron beam line 14BM, APS-BioCars, Argonne National
Laboratory, on an ADSC, CCD detector at a temperature of 100 K and an
energy of 12658 eV. Data from complexes, co-crystallized in the
presence of 0.2 mM EDTA, the 5 mM
Tl+ complex without EDTA, and the Mg2+ complex
without Tl+ and EDTA were collected on an R-AXIS
IV++/Rigaku rotating anode at a temperature of 100 K, using
CuK Structure Determination, Model Building, and
Refinement--
Crystals grown for this study are isomorphous to PDB
code 1CNQ (R-state) or 1EYI (T-state). Structure determinations were
initiated by molecular replacement using calculated phases from either
1CNQ or 1EYI, less ligands and water molecules. Electron density maps
were calculated using CNS (33). For structures reported here, the
anomalous data set was accepted only if it resulted in significant
anomalous difference density at the positions of sulfur and phosphorus
atoms. Modifications to structural models were done through XTALVIEW
(34). Models were refined against x-ray data using CNS with force
constants and parameters of stereochemistry from Engh and Huber (35).
Final cycles of refinement used SHELX (36) with restraints on bonds and
angle distances. The thermal parameter for Tl+ at a
specific site was fixed to the average of thermal parameters for atoms
of coordinating side chains. Occupancies of Tl+ were
refined with SHELX, and confirmed in XTALVIEW against
2Fobs Kinetic Experiments--
Assays employed the coupling enzymes,
phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (1). The
coupling enzymes were dialyzed exhaustively in order to remove
NH Purity of FBPase and the Influence of Tl+ on
Kinetics--
FBPase used here migrates as a single band on an
SDS-polyacrylamide gel and exhibits no evidence of proteolysis. A
previous report regarding Tl+-activation of FBPase from
mouse liver did not provide experimental details (38) and hence the
phenomenon was re-investigated here. Tl+ does not influence
the coupling enzymes of the assay system, so that the rate of formation
of NADH is directly related to the rate of formation of F6P. The
maximum level of Tl+-activation for the recombinant porcine
enzyme is 20%, with a Hill coefficient of 1.15 ± 0.09 and a
Ka of 1.3 ± 0.1 mM. (The
Ka for mouse liver enzyme is 16 mM). At
Tl+ concentrations in excess of 15 mM, the
specific activity of FBPase declines. Maximal K+-activation
for the recombinant porcine enzyme under comparable assay conditions is
18% (26), with a Hill coefficient of unity and a Ka
of 17 mM (13).
Quality of Crystals--
Conditions of crystallization of R-state
FBPase differ from those employed in past work (17, 20) in two
respects: (i) F6P2 replaces F6P/Pi, and (ii)
glycerol is present as a cryo-protectant. Co-crystallization with
substrate, rather than products, should not alter the results. The
enzyme is active under the conditions of crystallization, and thus
products and substrates should be at their equilibrium concentrations
regardless of the starting conditions. The addition of glycerol (27%,
v/v) and the reduced concentration of polyethylene glycol 3350 (from 10 to 8%, w/v), however, have resulted in an unexpected dividend.
Crystals, grown in the absence of glycerol, exhibit a wide variation in
x-ray diffraction properties after exposure to glycerol and rapid
freezing in liquid nitrogen. Crystals soaked in glycerol are fragile
and become disordered in 9 of every 10 instances. On the other hand, FBPase crystals grown in the presence of glycerol undergo rapid freezing with reproducible results. Under the new conditions of crystal
growth, R-state FBPase crystals can diffract to 1.3 Å resolution (44),
whereas previous crystals exhibit diffraction to 2.3 Å. The
co-crystallized Tl+ complexes reported below have a
resolution limit of 1.8 Å. The reduced resolution probably arises from
the combination of several distinct complexes within the same crystal
that differ in their sites of Tl+-association.
In all structures reported below, save the
Tl+/Mg2+/AMP/product complex, FBPase
crystallizes in the same space group (I222) and in isomorphous unit
cells (Table I). The enzyme is in its
R-state, with dynamic loop 52
Thallium has been chosen over potassium in this study in order to
detect metal binding at low occupancy. As noted above, K+
and Tl+ have comparable effects on the function of FBPase.
The choice of wavelength here optimizes anomalous scattering from
Tl+ without introducing an anomalous signal from
Mg2+. At an energy of 8040 eV ( Crystal Structure of the Mg2+/Product
Complex (PDB: 1NUZ)--
Data from improved crystals reveal
electron density at site 3 consistent with Mg2+ and a
coordinated water molecule (Fig. 1,
top). Asp68 and Glu97 along with two
oxygen atoms of Pi complete the inner coordination shell
(square pyramidal geometry) of site-3 Mg2+. When assigned
fractional occupancies of 0.5, thermal parameters for the
Mg2+ and the water molecule at site 3 match those of nearby
atoms of the active site. Fractional occupancies of 0.5 for residues 61 Crystal Structure of the Tl+(1
mM)/Mg2+/Product Complex
(PDB: 1NV0 (1NV4 with EDTA))--
The addition of
Tl+ to a concentration of 1 mM in
crystallization experiments, results only in modest differences
relative to the Tl+-free complex. The 1-OH group of F6P now
fractionally occupies two conformations, pointed toward the P-atom of
Pi and away from that atom. The later conformation for the
1-OH group was observed in the Zn2+/product complex (17,
20). Some anomalous difference density appears at sites 1 and 2, at
positions slightly displaced from density associated with
Mg2+ cations (Table I). The anomalous density may result
from the association of Tl+ to active sites having no
Pi. Tl+ cannot occupy sites 1 and 2 defined by
Mg2+ in the presence of Pi, because of
unacceptably short distances between oxygen atoms and the metal cation.
Mg2+ remains at site 3, and a small peak of anomalous
density appears at site 3a, described in detail in the following complex.
Crystal Structure of the Tl+(5
mM)/Mg2+/Product Complex
(PDB: 1NV1 (1NV5 with EDTA))--
At a concentration of 5 mM Tl+, strong anomalous density appears at
sites 3a, 3b, 4, and 5 (Fig. 1). Atoms that lie within 3 Å of the
thallous ions are in Table II. Site 3a
corresponds to site 3 of Villeret et al. (27).
Tl+ at site 3a (as for sites 3b, 4, and 5) is at low
occupancy (Table I). Furthermore, the fractional amount of the 1-OH
group, directed away from the P-atom of Pi, has increased
(Table I), and in fact Tl+ at site 3a could coordinate the
1-OH group of F6P. The binding of Tl+ to sites 3a and 3b
must be mutually exclusive, as their distance of separation is only 2.5 Å. No electron density is present for Mg2+ at site 3, and
segment 61 Crystal Structures of the Tl+(20 and 100 mM)/Mg2+/Product Complex
(PDB: 1NVZ (1NV6 with EDTA) and 1NV3, respectively)--
The
trend defined by the 1 and 5 mM Tl+ complexes
is essentially complete at a concentration of 20 mM
Tl+. Thallous cations at sites 3a, 3b, 4, and 5 do not
reach full occupancy, nor does a Tl+ concentration of 100 mM significantly increase cation levels at sites 3 Crystal Structure of the Tl+(20
mM)/Mg2+/AMP Product
Complex (PDB: 1NV7)--
Villeret et al. (27)
reported a Tl+(10 mM)/AMP complex with the
substrate analog 1,5-anhydroglucitol 6-phosphate. The complex reported
here differs in the substitution of products for an inhibitor and the
inclusion of Mg2+, an essential activator of FBPase. In the
complex of Villeret et al., Tl+ occupies sites
1, 2, and 3a, using our nomenclature for the metal sites in FBPase. In
our complex, Mg2+ is at site 1, whereas Tl+
occupies sites 2, 3a, 3b, and 4 (Fig. 1, bottom). Occupancy
factors for all Tl+ sites are ~0.15, but the
Mg2+ at site 1 is at full occupancy (Table I). We observed
Tl+ at sites 3b and 4, not observed by Villeret et
al. Reported differences in metal ligation undoubtedly arise from
differences in crystallization conditions (principally the presence or
absence of Mg2+), but as AMP-ligated FBPase is an
inhibited form of the enzyme, the functional relevance of either
structure is unclear.
The presence of Mg2+ at site 3 is correlated with the
appearance of ordered structure for segment 6120 mM) displace Mg2+ from
site 3 and the 1-OH group of fructose 6-phosphate from in-line geometry
with respect to bound orthophosphate. Loop 52
72 appears in a new
conformational state, differing from its engaged conformation by
disorder in residues 61
69. Tl+ does not bind to metal
sites 1 or 2 in the presence of Mg2+, but does bind to four
other sites with partial occupancy. Two of four Tl+
sites probably represent alternative binding sites for the site 3 catalytic Mg2+, whereas the other sites could play roles in
monovalent cation activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
72 evidently plays a central role in allosteric inhibition of
catalysis by AMP. It can be in at least three conformational states:
engaged, disordered, and disengaged (17, 20, 23). The engaged
conformation is arguably required for the high affinity association of
divalent cations and in stabilizing the transition state (17, 23-26).
The disordered conformation of the loop may facilitate product release
and substrate association. The loop can be in its engaged or disordered
conformations in the R-state enzyme. The T-state, however, favors a
single (disengaged) conformation for the loop, which cannot stabilize
divalent cation association at the active site. Interactions between
the loop and residues near the N terminus of an adjacent subunit play
an important role in stabilizing the disengaged conformation of the
loop (23).
72. Mutations of specific residues of the dynamic loop
increase the Ka for Mg2+, and without
exception also abolish K+-induced effects on catalysis (25,
26). On the other hand, K+ does not increase the fraction
of subunits with an engaged loop in the presence of saturating
Mg2+/F6P/Pi (26). Hence, improved catalysis
comes from a more stable transition state in the presence of
K+.
3.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
radiation, passed through an Osmic confocal mirror system. Data
from synchrotron sources were reduced and scaled by Denzo/Scalepack
(31). Data from the R-AXIS IV++ were processed with
CrystalClear (32).
Fcalc omit maps
and anomalous difference maps.
. The concentration of F16P2 in all assays
was 20 µM. The reduction of NADP+ to NADPH
was monitored by fluorescence emission at 470 nm, using an excitation
wavelength of 340 nm, as described elsewhere (23). Concentrations of
Tl+ were 0.0, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, 8.0, and 10.0 mM. All kinetic assays were performed at room temperature
in triplicate. Initial rate data were fit using ENZFITTER (37). The
Hill coefficient for Tl+ activation came from a least
squares fit of the following shown in Equation 1,
where V is the observed initial velocity at a
specific concentrations of Tl+, S is the
concentration of Tl+, n is the Hill coefficient,
Ka is the affinity constant for Tl+, and
3.0863 is the initial velocity of the reaction in the absence of
Tl+, based upon an average of five determinations.
(Eq. 1)
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
72 in an engaged conformation, as
defined previously by Zn2+/product complexes (17, 20). The
side chain of Tyr57 in the engaged conformation occupies a
hydrophobic pocket (26). We observed no significant differences in the
presence or absence of 0.2 mM EDTA. In the
Tl+/Mg2+/AMP/product complex, FBPase adopts a
T-state global conformation, with a disengaged loop 52
72 (20). As
these complexes have been reported in detail in prior publications (17,
20), we focus here on changes in the active site in response to
different conditions of crystal growth.
Statistics of data collection and refinement
= 1.524 Å),
thallium and magnesium have f' of
4.03 and 0.172 electrons,
respectively, and f" of 8.12 and 0.177 electrons, respectively. At
an energy of 12658 eV (
= 0.979 Å), thallium and magnesium
have f' of
18.9 and 0.0876 electrons, respectively, and f" of
3.93 and 0.0714 electrons, respectively (41). (f' and f" are
the real and imaginary components of anomalous scattering). On the
basis of the above, magnesium contributes virtually nothing to
anomalous scattering, and in fact no anomalous difference density
appears at the metal-binding loci of Mg2+ complexes, even
though distinct anomalous difference density appears at sulfur atoms
(data not shown). Thallous ions bound at low fractional
occupancy (0.1, for instance) may be mistaken for water
molecules in electron density maps, but can be identified unambiguously
in an anomalous difference map. Finally, if Mg2+ and
Tl+ mutually exclude each other at a binding site, then
anomalous scattering data allows a direct estimate of the
Tl+ occupancy, and an indirect estimate of the
Mg2+ occupancy at that site. Hence, the anomalous data
eliminates much ambiguity in the interpretation of electron density
associated with possible metal sites, and as presented below, reveals a
far more complex set of interactions than had been suggested by
previous studies (27).
69 of dynamic loop 52
72 also result in thermal parameters that
match those for other atoms of the active site. The water molecule
coordinated to site-3 Mg2+ hydrogen bonds with
the side chain of Glu98 and is close to the side chain of
Asp74. Magnesium cations at sites 2 and 3 in combination
with their coordinated water molecules, Pi,
Asp74, Glu97, and Glu98, define an
interconnected assembly of atoms with well defined geometry (Fig. 1).
The 1-OH group of F6P coordinates the Mg2+ at site 1 and is
in contact with the P atom of Pi (distance of separation
approximately, 2.8 Å). Furthermore, the 1-O atom of F6P is equidistant
(approximately, 2.7 Å) from three oxygen atoms of Pi. The
angle defined by the 1-O atom of F6P, the P atom of Pi, and
the remaining (distal) oxygen atom of Pi is 172°. The distal oxygen atom of Pi coordinates to Mg2+ at
sites 2 and 3. The spatial relationships between the 1-OH group of F6P,
the Mg2+ at site 1, and bound Pi are
essentially identical to those reported in the previous
Mg2+/product complex (20). The Mg2+ at site 3 and its coordinated water molecule are probably important to the
catalytic mechanism of FBPase, as discussed below.
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Fig. 1.
Stereoview of electron density associated
with metal cations in the active site of FBPase. The
Mg2+(5 mM) complex has density from a
2Fobs Fcalc map
contoured in blue at 1
with a cutoff radius of 1 Å (top). The Mg2+(5
mM)/Tl+(20 mM) complex has density
from a 2Fobs
Fcalc map
contoured in blue at 1
with a cutoff radius of 1 Å and density from
an anomalous difference map contoured in red at 4
with a cutoff
radius of 1 Å (middle). The Mg2+(5
mM)/Tl+(20 mM)/AMP(5
mM) complex has density from a
2Fobs
Fcalc map
contoured in blue at 2
with a cutoff radius of 1 Å and density from
an anomalous difference map contoured in red at 4
with a
cutoff radius of 1 Å (bottom). MOLSCRIPT (40) and RASTER3D
(39) were used in the preparation of this illustration.
69 is less ordered than in the 1 mM Tl+ complex, as evidenced by increased thermal parameters
(Table I). Tl+ at sites 3a and 3b have displaced
Arg276 from the active site. Tl+ at site 4 occupies the position of the water molecule, coordinated to the site-3
Mg2+ in the Tl+-free complex. Tl+
at site 5, however, coordinates the side chains of Ser123
and Asp74 without the displacement of Mg2+ and
its coordinated water molecule from site 2. The oxygen atom of
Pi, distal with respect to the 1-OH group of F6P, is 3.4 and 3.1 Å from thallous ions at sites 4 and 5, respectively, ~0.5 Å beyond that generally observed for inner-sphere coordination of
Tl+ (42).
Metal site coordination
5
(Table I). In fact, at 100 mM Tl+ no metal
cation occupies site 1 (Mg2+ is now absent). In the 20 mM Tl+ complex, electron density is absent for
segment 61
69, the 1-OH group of F6P is directed entirely away from
the P-atom of Pi, and the side chain of Arg276
is displaced from the active site.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
69 of the dynamic loop 52
72. The effect of Mg2+ at site 3 is consistent with an
ordered loop in complexes that have Zn2+ a full occupancy
at metal site 3 (17, 20). Residues 61
69 define a reverse-turn segment
that lies immediately over the active site (Fig.
2). Evidently, loop 52
72 can remain in
the engaged conformation (Tyr57 can remain in its
hydrophobic pocket), while residues 61
69 cycle between a conformation
that stabilizes Mg2+ at site 3 (by coordination to
Asp68) and other conformations that allow ligand exchange
between the active site and bulk solvent. Mutations of
Asp68 cause the Ka for Mg2+
to rise 30-fold,2 corroborating
the putative role ascribed to Asp68 as a chelator of a
catalytically important Mg2+. Hence, the transition of loop
52
72 between engaged, disordered and disengaged conformations is
probably related to allosteric inhibition by AMP, whereas mobility in
segment 61
69 may be important to catalytic turnover in an R-state
enzyme with an engaged-loop.
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Fig. 2.
A new conformational state for loop
52 72. The FBPase tetramer (top)
showing the R-state, engaged loop conformation. Residues 61
69, which
become disordered with increasing concentrations of Tl+,
are in black. Gray spheres represent F6P
molecules. Active sites and ligands are not shown on the face of the
tetramer hidden from view. A more detailed view (bottom)
showing the proximity of residues 61
69 (main-chain ribbon in
black) to metal binding site 3 (M3),
Pi, and F6P. This illustration was drawn with MOLSCRIPT
(40).
The Mg2+ complex is consistent with an associative pathway
for the hydrolysis of F16P2 (Fig.
3). Magnesium ions at sites 2 and 3 coordinate the distal oxygen of Pi (the oxygen atom to be
displaced by the in-line attack of the 1-OH group of F6P). Each of
these magnesium cations also coordinates a water molecule, either of which could provide a proton to neutralize the negative charge on
leaving oxygen atom. Glu98 is a proton acceptor in hydrogen
bonds with both of these water molecules. Glu98 is required
for catalysis; mutations at position 98 cause a 10,000-fold decrease in
activity under conditions that support full activity of the wild-type
enzyme (43). Asp74, another residue essential for catalysis
(24), hydrogen bonds only with the water molecule coordinated to
Mg2+ at site 2. In fact, the oxygen atom of
Asp74 nearest to the site-3 metal may be protonated (Fig.
3). The hydrogen atom on the 1-OH group of F6P (coordinated to site-1
Mg2+) moves to an oxygen atom of the phosphate (3 oxygen
atoms of Pi are all 2.7 Å from the 1-O atom of F6P). The
oxygen of Pi, which hydrogen bonds with backbone amides 122 and 123, may be the most likely acceptor for the proton transferred
from the 1-OH group of F6P.
|
Although the catalytic mechanism of Fig. 3 assumes an associative
pathway for the hydrolysis of a phosphate ester, structures at near
atomic resolution support the existence of metaphosphate in the active
site of FBPase (44). The dissociative pathway for phosphoryl hydrolysis
then is a distinct possibility, but the roles of catalytically
essential side chains and the metal cations at sites 13 remain the
same regardless of pathway.
The kinetics of monovalent cation activation is generally consistent
with a simple mechanism, whereby the cation binds to a site distinct
from those of the divalent cations (27, 13). Kinetic data, however,
cannot exclude more complicated mechanisms, and the structures above
reveal the potential for a very complex mechanism of monovalent cation
activation. On the surface, the structures here agree well with the
kinetics. The Hill coefficient for Tl+-activation is 1.15, and the sum over occupancy factors for Tl+ at sites 35 is
1.2. The Ka for Tl+-activation is 1.34 mM and the sum over occupancy factors for Tl+
sites of the 5 mM complex is approximately one-half of that
at saturating levels of Tl+. The agreement between
structure and kinetics, however, may be only a coincidence. The
crystallographic work here measures the binding of Tl+ to
an active site ligated by F6P and Pi, whereas the enzyme
assay probes the influence of Tl+ on a
F16P2-ligated active site. To the best of our knowledge, there are no reports in the literature concerning the effects of
monovalent cations on the reverse reaction catalyzed by FBPase.
Each crystalline complex here represents an average of two or more states of metal ligation. The pure Mg2+ complex is a mixture of active sites with and without Mg2+ at site 3. In the Tl+(20 mM)/Mg2+ complex, Mg2+ at site 3 and Tl+ at sites 3b and 4 are mutually exclusive, Tl+ at sites 3a and 3b are mutually exclusive, and metal sites 3, 3a, 3b, 4, and 5 are mutually antagonistic (occupancy factors sum to near unity; Table I). As evidenced by the Tl+(100 mM)/Mg2+ complex, metal site 1 and metal sites 3a, 3b, 4, and 5 could be antagonistic as well, and in fact, inhibition by elevated concentrations of Tl+ may arise from the displacement of Mg2+ from site 1.
This averaging effect in the crystal obscures the functional
significance of each Tl+ binding site, but data from
Mg2+ complexes of FBPase at near atomic resolution provides
some important insights (44). Firstly, metaphosphate/hydroxide appears
in the active site of FBPase whenever the 1-OH group of F6P rotates
away from its in-line orientation. We observe here the rotation of the
1-OH group away from an in-line orientation as the occupancy of
Tl+ increases at site 3a. In fact, Tl+ at site
3a coordinates the 1-OH group and may be more effective than
Arg276 in stabilizing the formation of metaphosphate in a
dissociative reaction pathway. Hence, as originally suggested by
Lipscomb and co-workers (27), site 3a could be a site of
monovalent cation activation. On the basis of near atomic resolution
data Tl+ at site 4 coincides with an alternative binding
site for Mg2+. In the initial F16P2 complex,
Mg2+ may bind to site 4 and then migrate to site 3 after
the formation of metaphosphate (44). Tl+ association at
site 4 may be inhibitory because of its displacement of
Mg2+, or Tl+ at sites 4 and 3a together may
support catalysis more effectively than Mg2+ at site 3 along with Arg276 at site 3a. Tl+ at site 5 may
not interfere with the binding of Mg2+ to sites 13. Each
of the lone pair orbitals of the distal (leaving) oxygen atom of
Pi, could coordinate a metal cation (Mg2+ at
sites 2 and 3, and Tl+ at site 5), so that an FBPase
complex of Mg2+(sites 1
3)/Tl+(site 5) may be
active and may exhibit enhanced catalysis.
Studies here have identified three new loci (sites 3b, 4, and 5) at the
active site of FBPase for cation association in the presence of
Mg2+. These new sites are all within coordination distance
of the substrates/products and/or amino acid residues essential to
catalysis. Together these sites suggest the possibility of different
pathways for the hydrolysis of F16P2. Under in
vivo conditions, however, where FBPase can select from a multitude
of cations (Mg2+, Mn2+, Zn2+,
Ca2+, K+, and Li+) at physiological
concentrations and combine these cations together with products or
substrate, all cation binding sites revealed here could be important in
determining the net flux of reactants through gluconeogenesis.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Research Grant NS-10546 and National Science Foundation Grants MCB-9985565.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NUZ, 1NV0, 1NV1, 1NV2, 1NV3, 1NV4, 1NV5, 1NV6, and 1NV7) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.: 515-294-6116;
Fax: 515-294-0453; E-mail: honzatko@iastate.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212394200
2 C. Iancu, H. J. Fromm, and R. B. Honzatko, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; F6P, fructose 6-phosphate, F16P2, fructose 1,6-bisphosphate; F26P2, fructose 2,6-bisphosphate; PDB, protein data bank.
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