From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011
Received for publication, December 5, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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The hydrolysis of a phosphate ester can proceed
through an intermediate of metaphosphate (dissociative mechanism) or
through a trigonal bipryamidal transition state (associative
mechanism). Model systems in solution support the dissociative pathway,
whereas most enzymologists favor an associative mechanism for
enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase
grow from an equilibrium mixture of substrates and products at near
atomic resolution (1.3 Å). At neutral pH, products of the reaction
(orthophosphate and fructose 6-phosphate) bind to the active site in a
manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K+ (200 mM), or at pH 9.6, metaphosphate and water (or
OH Phosphatases, mutases, kinases, and nucleases all catalyze
phosphoryl transfer reactions central to biochemical processes that
sustain life. The transfer of the phosphoryl group of a phosphate ester
to water in most cases requires metal ions as cofactors, and proceeds
either by way of a trigonal-bipyramidal transition state (associative
mechanism), or through the formation of an unstable intermediate of
metaphosphate (dissociative mechanism) (1 Fructose-1,6-bisphosphatase
(D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC
3.1.3.11, hereafter FBPase)1
is a key regulatory enzyme in gluconeogenesis. FBPase catalyzes the
hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose 6-phosphate (F6P) and orthophosphate (Pi). The inhibition
of FBPase in mammals results in reduced levels of serum glucose in the
fasting state. Hence, FBPase is a target for the development of drugs in the treatment of non-insulin dependent diabetes, which afflicts over
15 million people in the United States (16, 17).
FBPase can be in either of two quaternary conformations, the R-state
(catalytically active) or the T-state (inactive) (18). AMP and fructose
2,6-bisphosphate both inhibit catalysis by FBPase, the former through
an allosteric mechanism (19, 20) and the latter by direct ligation of
the active site (21, 22). Divalent metals (Mg2+,
Mn2+, or Zn2+) are essential for FBPase
activity. Monovalent metals (K+, Rb+,
Tl+, or NH3+) further enhance
reaction rates at relatively low concentrations, but can be inhibitory
at high concentrations (23 FBPase crystallizes readily from an equilibrium mixture of products and
substrates, and in fact the enzyme itself is active under conditions of
crystallization. In past crystal structures of FBPase, only
orthophosphate and F6P have appeared in the active site (29, 30).
Presumably, the observed complexes represent a minimum free energy
under the conditions of the crystallization experiment. Reported here
are crystal structures of FBPase at near atomic resolution, in which a
partial reaction (essentially the second step of a dissociative
pathway) has lead to the formation of metaphosphate in the active site.
The crystallographic structures do not constitute irrefutable evidence
of a dissociative reaction pathway, but they do demonstrate that FBPase
can generate and stabilize metaphosphate at its active site. Hence,
variations observed in the mechanism of catalysis by FBPase may arise
from changes in the rate-limiting step of a dissociative pathway or even a change in pathway, for instance, from associative to dissociative.
Protein Isolation and Crystallization--
FBPase was isolated
as described previously (29 Data Collection--
Data from the low K+ complex
(control complex) were collected at APS-Structural Biology Center (beam
line 19BM), Argonne National Laboratory, on an SBC-CCD at 100 K, using
a wavelength of 1.0 Å. Data from the high K+ complex were
collected at the synchrotron beam line X4A, Brookhaven National
Laboratory, on an ADSC Quantum4 CCD at 100 K, using a wavelength of
0.9795 Å. Data from the high pH complex were collected at APS-BioCars
(beam line 14BM), Argonne National Laboratory on an ADSC Quantum4 CCD
at 100 K, using a wavelength of 1.0 Å. Data from synchrotron sources
were reduced and scaled by Denzo/Scalepack (32).
Structure Determination and Refinement--
Crystals grown for
this study are isomorphous to PDB code 1EYJ. Structure determinations
were initiated by molecular replacement using calculated phases of
1EYJ, and then refined by SHELX (33). Model building and modifications
employed XTALVIEW (34). Only distance restraints between covalently
link atoms were applied to orthophosphate and metaphosphate, allowing
structure factors to be the principal determinant of the geometry of
the phosphoryl groups in each of the complexes. Fractional occupancy
factors for a given atom or group of atoms were determined by
refinement of thermal parameters for a series of fixed occupancy
factors. Reported here are occupancy factors that resulted in refined
thermal parameters comparable to those of neighboring atoms in full occupancy.
Three complexes (Table I) are
presented here: (i) orthophosphate, F6P, Mg2+, and
K+ (10 mM) at pH 7 (hereafter the control
complex), (ii) metaphosphate, F6P, Mg2+, and K+
(200 mM) at pH 7 (hereafter the high K+
complex), and (iii) metaphosphate, F6P, and Mg2+ at pH 9.6 (hereafter the high pH complex). All complexes have the dynamic loop
(residues 52) are in equilibrium with orthophosphate. Furthermore,
one of the magnesium cations in the pH 9.6 complex resides in an
alternative position, and suggests the possibility of metal cation
migration as the 1-phosphoryl group of the substrate undergoes
hydrolysis. To the best of our knowledge, the crystal structures
reported here represent the first direct observation of metaphosphate
in a condensed phase and may provide the structural basis for
fundamental changes in the catalytic mechanism of
fructose-1,6-bisphosphatase in response to pH and different metal
cation activators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6). The metaphosphate anion
(PO
15). The likelihood of trapping metaphosphate in the active site of
an enzyme is remote, because of the proximity of acceptor molecules in
the active site. Yet an enzyme offers an advantage in that it reduces
the free energy of the transition state. Hence, the active site itself could serve as a thermodynamic trap if metaphosphate, once generated, is denied access to an acceptor.
25). The enzyme-mediated reaction is
pH-dependent; plots of initial velocity versus
Mg2+ are sigmoidal (Hill coefficient of 2) at neutral pH,
but hyperbolic at pH 9.6 (26). The kinetic mechanism at pH 7 with
Mg2+ as the cation activator is steady-state random (25),
whereas at pH 9.6 the kinetic mechanism is rapid-equilibrium random
(26). The catalytic mechanism is also sensitive to the type of cation activator: The Mn2+-activated enzyme uses exclusively the
-anomer of F16P2 (27), but Mg2+-activated
FBPase uses both
- and
-anomers of F16P2 (23). Mn2+-activated FBPase, but not the
Mg2+-activated enzyme, hydrolyzes the substrate analogue
(Sp)-[1-18O]fructose 1-phosphothioate
6-phosphate (28). The zinc cation is an activator of FBPase, and yet
traces of Zn2+ reduce catalytic rates of the
Mg2+-activated enzyme (24). The above suggests alternative
catalytic pathways, the dominant mechanism being determined by pH, the
kind of cation-activator, and the conformation of the substrate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31). Crystals in 10 or 200 mM
K+ grew (by the method of hanging drops) from equal parts
of a protein solution (10 mg/ml FBPase, 50 mM Hepes, 5 mM MgCl2, 5 mM F16P2, 10 mM or 200 mM KCl) and a precipitant solution
(100 mM Hepes pH 7, 5% t-butyl alcohol, 27 or
21% (v/v) of glycerol, and 8% (for 10 mM KCl) or 14%
(for 200 mM KCl) (w/v) polyethylene glycol 3350). Crystals
at pH 9.6 grew from equal parts of a protein solution (10 mg/ml FBPase,
10 mM KPi, 5 mM MgCl2,
5 mM F16P2), and a precipitant solution (100 mM glycine, pH 9.6, 5% t-butyl alcohol, 25%
(v/v) glycerol, and 10% (w/v) polyethylene glycol 3350). The droplet volume was 4 µl. Wells contained 500 µl of the precipitant
solution. Crystals of uniform dimension (0.2 mm) grew in three to 5 days at room temperature. All crystals belong to space group I222, with
one subunit of FBPase in the asymmetric unit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
72) in its engaged conformation, as determined by the
location of Tyr57 (29, 30). Conditions that result in the
formation of metaphosphate differ only in pH (7 versus 9.6)
or the concentration of K+ (10 versus 200 mM). The kinetic mechanism of FBPase changes from steady-state random at pH 7 to rapid-equilibrium random at pH 9.6 (26),
and K+ is inhibitory at concentrations of 200 mM (Ki of 68 mM), but
activating at concentrations of 10 mM
(Ka of 17 mM) (25).
Statistics of data collection and refinement
Control Complex (PDB: 1NUY)--
Aside from its substantially
higher resolution (Table I), the control complex is essentially
identical to the Mg2+ product complex of Choe et
al. (29, 30, 40). Orthophosphate is clearly at the active site,
and Mg2+ occupies sites 1, 2, and 3 (Fig.
1A). The 1-OH group of F6P is in two positions, related by a rotation of approximately of 120° about the C1C2 bond axis. In one of its positions (occupancy factor
of 0.7), the 1-OH group coordinates the Mg2+ at site 1, optimally positioned for an associative reaction (Fig. 2A). Three of four oxygen
atoms from Pi are approximately equidistant from the 1-OH
group of F6P and the distance between the phosphorus atom and the
oxygen atom of the 1-OH group is 2.72 Å. In its second position
(displaced conformation; occupancy factor of 0.3), the 1-OH group is
turned away from in-line geometry, hydrogen bonding with only one of
the oxygen atoms of Pi (Fig. 2A).
Mg2+ at site 1 is 4
5 coordinated (the 1-OH group of F6P
being responsible for the variation), whereas magnesium cations at
sites 2 and 3 are both six-coordinated (Fig. 2A).
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High K+ Complex (PDB: 1NUX)--
An increase in the
concentration of K+ from 10 to 200 mM results
in an anomalous signal at metal site 1 (Fig. 1B), indicating the displacement of the Mg2+ from that site by a different
atom type. The metal at site 1 has four inner-sphere ligands: One
oxygen atom each from Asp118, Aspl21,
Glu280, and the phosphoryl species is ~22.5 Å from
metal site 1 (Fig. 2B). The 1-OH group of F6P no longer
coordinates the site-1 metal, being entirely in its displaced
conformation. The anomalous signal from metal site 1 may be due to
K+ or a trace contamination of a transition series cation
(Zn2+, for instance) in the KCl. The latter is the most
likely cause. The inclusion of 0.2 mM EDTA in the
crystallization solution eliminates the anomalous signal at metal site
1. Furthermore, the tetrahedral coordination of the site-1 cation and
the displaced conformation of the 1-OH group of F6P are characteristics
of previously determined Zn2+-product complexes of FBPase
(29, 30). A mixture of Zn2+ and Mg2+ at
occupancies of 0.25 and 0.75, respectively, account for the magnitude
of the anomalous signal, and provide thermal parameters of ~24
Å2, equivalent to that of the site-1 Mg2+ in
the control structure
The thermal parameter associated with the Mg2+ at site 3 is higher than that of its counterpart in the control complex (30 versus 26 Å2). The Mg2+ cation may not fully occupy site 3. (As defined more clearly in the high pH complex below, Mg2+ could occupy a site near Glu98 at low occupancy, and not be resolved from the electron density associated with the water molecule that hydrogen bonds with Glu98 and coordinates the Mg2+ at site 3. See Fig. 2, B and C.) Further indications of weakened interactions involving Mg2+ at site 3 are an increase in the coordination distance to the oxygen atom of the phosphoryl species and the concomitant decrease in the donor-acceptor distance to Arg276 of that same oxygen atom (Fig. 2B).
The electron density associated with the ligand at the 1-phosphoryl
pocket is a distorted tetrahedron. An elongated teardrop of electron
density extends from a plane of electron density. Metaphosphate fits
the planar density well, and a water molecule (perhaps representing a
molecule of hydroxide) fits equally well to the teardrop of electron
density. Thermal parameters of the metaphosphate molecule and its
associated water/hydroxide molecule, both refined at full occupancy,
are comparable to those of the ligating Mg2+ and nearby
side chains. The water molecule is 2.35 Å away from the phosphorus
atom of metaphosphate, being coordinated to the magnesium cations at
sites 2 and 3, and is on the verge of hydrogen bonding to the side
chain of Asp74 (Fig. 2B). The electron density
probably represents an equilibrium mixture of orthophosphate and
PO.
High pH Complex (PDB: 1NUW)--
Glu97, which in the
control and high K+ complexes coordinates magnesium cations
at sites 2 and 3, now bridges the magnesium cations at sites 1 and 2. The Mg2+ at site 1 is at least 5-coordinated and the
Mg2+ at site 2 remains six-coordinated (Figs. 1C
and 2C). As in the high K+ complex, the 1-OH
group of F6P is in its displaced orientation. The loss of
Glu97 as a coordinating ligand to Mg2+ at site
3 is linked perhaps to the binding of magnesium cations at sites 3 and
4. A similar conformational change in Glu97 occurs in
Mg2+/Tl complexes, in which Tl+ at site 4 displaces Mg2+ at site 3 (40). Glu98
coordinates to Mg2+ at site 4, whereas Asp68
coordinates to Mg2+ at site 3, but the binding of metal
cations to sites 3 and 4 are probably mutually exclusive, as they are
only 2.5 Å apart. Thermal parameters for Mg2+ at sites 3 and 4 are 23 and 21 Å2, respectively, with fractional
occupancies of 0.6 and 0.3. The electron density at the 1-phosphoryl
pocket appears as an elongated teardrop extending from a plane (Fig.
1C). A molecule of metaphosphate, distorted from planarity,
and a water molecule (or hydroxide anion) provide the best fit to the
electron density. The oxygen atom of the water molecule is 3.09 Å from
the phosphorus atom of the metaphosphate, and is within the
coordination spheres of the magnesium cations at sites 2 and 4 (or 3),
and within hydrogen bonding distance of Asp74 (Fig.
2C). As in the high K+ structure, the electron
density at the 1-phosphoryl pocket is consistent with an equilibrium
mixture of orthophosphate and
PO.
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DISCUSSION |
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The control complex may represent a step on the reaction pathway, but it almost certainly does not represent the central kinetic complex (the interconversion of F16P2 and F6P/Pi at the active site). Liu and Fromm (22) determined a value of 2 for the equilibrium constant of the central kinetic complex. The electron density is consistent, however, with the presence of only F6P and orthophosphate. Conditions of crystallization and packing interactions of the crystal could perturb the equilibrium constant of the central complex in favor of products, or the crystallographic complex itself could represent a dead-end complex not on the reaction pathway. Nonetheless, the geometric relationship between Pi and F6P (Fig. 2A) is consistent with an associative reaction mechanism, the details of which have been presented elsewhere (40); Asp74 (the pKa of which is raised by the proximity of Glu98) abstracts a proton from a water molecule coordinated to the Mg2+ at site 2 or site 3. The resulting Mg2+-coordinated hydroxide anion in turn abstracts the proton from a second water molecule (the attacking nucleophile) that bridges the magnesium cations at sites 2 and 3. The attacking hydroxide anion has in-line geometry with respect to the 1-phosphoryl group, and would be less than 3 Å from the P-1 atom. Data in support of catalytic roles for Asp74 and Glu98 come from directed mutations, which cause more than a 10,000-fold reduction in catalytic rates (31, 36). The associative pathway must have a double proton transfer, because the catalytic base (Asp74/Glu98 subassembly) is too far from the water molecule bridging the magnesium cations at sites 2 and 3 for a direct hydrogen bond.
The arrangement of catalytic side chains and ligands in the active site
of FBPase, however, are also consistent with a dissociative pathway,
and indeed FBPase can generate metaphosphate and the hydroxide anion
from orthophosphate. To the best of our knowledge, the high pH and high
K+ complexes reported here are the first direct
observations of metaphosphate in a condensed phase. FBPase in its
crystalline complex catalyzes the second step of a dissociative
mechanism; however, the formation of F16P2 from
PO
The presence of Mg2+ cations at mutually exclusive loci
(sites 3 and 4) and the alternative ligation of cation sites by
Glu97 suggest the possibility of significant change in the
active site during the course of the reaction. The Mg2+ at
site 4, a cation-binding site identified in
Mg2+/Tl+ complexes of FBPase (40), may in fact
play a direct role in catalysis (Fig. 3).
In F16P2 complexes of FBPase, Mg2+ may appear
initially at sites 1, 2, and 4. Stereoinversion of the 1-phosphoryl
group may favor the migration of Mg2+ from site 4 to 3. Hence, in all crystalline complexes of Pi and F6P,
Mg2+ (or Zn2+) is at site 3, whereas in
F16P2 complexes the preferred binding site for
Mg2+ may be site 4. Asp74 then could activate a
water molecule coordinated to magnesium cations at sites 2 and 4 for a
nucleophilic attack on the metaphosphate anion (Fig. 3).
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A change in metal cation coordination by Glu97 could facilitate the putative migration of Mg2+ from site 4 to 3 during catalysis. In a dissociative pathway, charge builds on the O-1 atom of F16P2, and as a consequence the Mg2+ at site 1 may release Glu97 from its crowded coordination sphere. A modest conformational change puts Glu97 into the nascent coordination sphere of metal site 3, which in turn promotes the migration of the cation (with its attached hydroxide anion) from site 4 to 3 (Fig. 3). The migration of Mg2+ between sites 3 and 4 would account for the significance of both Asp68 (which coordinates Mg2+ at site 3) and Glu98 (which coordinates Mg2+ at site 4) in catalysis. Mutations2 of Glu98 (36) and Asp68 each reduce catalytic rates of FBPase by orders of magnitude under comparable conditions of assay. The dissociative pathway, as in Fig. 3, replaces the double proton transfer of the associative pathway, with the migration of a metal-bound hydroxide anion.
The loss of Mg2+ cooperativity at pH 9.6 in FBPase kinetics may simply reflect a change in the rate-limiting step of a dissociative pathway. Mutations of Asp68 eliminate Mg2+ cooperativity at pH 72 and thereby implicate the cation in site 3 in cooperative phenomenon. The generation of the hydroxide anion at pH 7 may require the participation of Asp74 and metal cations at sites 2 and 4 (or 3), whereas at pH 9.6 the generation of the hydroxide anion may occur with less involvement from the active site. Hence, the formation of metaphosphate may be limiting at pH 9.6, whereas the formation of hydroxide anion may be limiting at pH 7.
The hydrolysis of most phosphate esters in organic model systems occurs
by a dissociative mechanism (36), and the same chemistry may occur in
the active site of FBPase. In fact, Herschlag and Jencks (37) present a
scheme (Chart II or III) that is strikingly similar to the complexes
reported here in the relative placement of Mg2+, a
metal-coordinated hydroxide ion and a planar intermediate. Furthermore,
there are striking parallels between the active sites of FBPase and
alkaline phosphatase (38), suggesting that the chemistry of model
systems is broadly applicable to phosphatases. Ancestral phosphatases
long ago may have commandeered the dominant reaction pathway in
solution, but through evolution the free-energy landscape of the
reaction coordinate may now place the associative and dissociative
pathways on a near equal footing. Hence, small perturbations in the
relative free energies of transition states, due to changes in metal
cofactors, pH, chemical composition of the substrate, and/or conditions
of crystallization may leverage profound effects on the catalytic mechanism.
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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 Grant 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 1NUX, 1NUY, and 1NUW) 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.: 515-294-7103;
Fax: 515-294-0453; E-mail: honzatko@iastate.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212395200
2 C. V. Inacu and R. B. Honzatko, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; F16P2, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate.
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