Interaction of Tl+ with Product Complexes of Fructose-1,6-bisphosphatase*

Jun-Yong Choe, Scott W. Nelson, Herbert J. Fromm, and Richard B. HonzatkoDagger

From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011

Received for publication, December 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>4</SUB><SUP>+</SUP></UP>) will further enhance enzyme activity. Here, the interaction of Tl+ with fructose-1,6-bisphosphatase is explored under conditions that support catalysis. On the basis of initial velocity kinetics, Tl+ enhances catalysis by 20% with a Ka of 1.3 mM and a Hill coefficient near unity. Crystal structures of enzyme complexes with Mg2+, Tl+, and reaction products, in which the concentration of Tl+ is 1 mM or less, reveal Mg2+ at metal sites 1, 2, and 3 of the active site, but little or no bound Tl+. Intermediate concentrations of Tl+ (5-20 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 52-72 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).

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 52-72. 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+.

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 1-3.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 CuKalpha 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).

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-Fcalc omit maps and anomalous difference maps.

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<UP><SUB>4</SUB><SUP>+</SUP></UP>. Thallium acetate solutions were prepared immediately prior to their use in assays. Tl+ up to a concentration of 15 mM had no effect on the coupling enzymes. Assays were initiated by the addition of magnesium acetate (final concentration of 5 mM), instead of magnesium chloride, to avoid the precipitation of Tl+ by Cl-. 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,


V=[V<SUB>m</SUB>S<SUP>n</SUP>/(K<SUB>a</SUB>+S<SUP>n</SUP>)]+3.0863 (Eq. 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 52-72 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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Statistics of data collection and refinement
Mg2+ is present in all complexes at 5 mM.

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 (lambda  = 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 (lambda  = 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).

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 61-69 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.


View larger version (34K):
[in this window]
[in a new window]
 
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 1sigma 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 1sigma with a cutoff radius of 1 Å and density from an anomalous difference map contoured in red at 4sigma 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 2sigma with a cutoff radius of 1 Å and density from an anomalous difference map contoured in red at 4sigma with a cutoff radius of 1 Å (bottom). MOLSCRIPT (40) and RASTER3D (39) were used in the preparation of this illustration.

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 61-69 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).


                              
View this table:
[in this window]
[in a new window]
 
Table II
Metal site coordination
Listed are atoms within 2.5 Å of Mg2+ and 3.0 Å of T1+.

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 3-5 (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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The presence of Mg2+ at site 3 is correlated with the appearance of ordered structure for segment 61-69 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.


View larger version (30K):
[in this window]
[in a new window]
 
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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Associative mechanism for the reverse reaction of FBPase. Glu280, which coordinates to metal M1, and Glu97, which coordinates metals M2 and M3, come from above the plane of the schematic, and are not shown here for clarity. Thin, solid lines are coordinate bonds, dotted lines represent hydrogen bonds, and dashed lines represent partial covalent bonds. A, initial product complex. The proton on Asp74 is hypothetical. The 1-O atom of F6P (coordinated to M1) is the attacking nucleophile. An oxygen atom of orthophosphate abstracts the proton from the 1-hydroxyl group of F6P. B, transition state. The leaving oxygen atom abstracts a proton from the water molecule coordinated to M3. That same water molecule in turn accepts a proton from Asp74. C, penultimate complex. Transfer of the proton from the 1-phosphoryl group to the hydroxide anion, bridging M2 and M3, generates F16P2 and water.

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 1-3 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 3-5 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 1-3. 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.

    FOOTNOTES

* 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/).

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Krebs, H. A. (1963) in Advances in Enzyme Regulation (Weber, G., ed), Vol. 1 , pp. 385-400, Pergamon Press Ltd., London
2. Marcus, F. (1981) in The Regulation of Carbohydrate Formation and Utilization in Mammals (Veneziabe, C. M., ed) , pp. 269-290, University Park Press, Baltimore
3. Benkovic, S. J., and de Maine, M. M. (1982) Adv. Enzymol. Relat. Areas Mol. Biol. 53, 45-82[Medline] [Order article via Infotrieve]
4. Hers, H. G., and Hue, L. (1983) Annu. Rev. Biochem. 52, 617-653[CrossRef][Medline] [Order article via Infotrieve]
5. Tejwani, G. A. (1983) Adv. Enzymol. Relat. Areas Mol. Biol. 54, 121-194[Medline] [Order article via Infotrieve]
6. Pilkus, S. J., El-Maghrabi, M. R., and Claus, T. H. (1988) Annu. Rev. Biochem. 57, 755-783[CrossRef][Medline] [Order article via Infotrieve]
7. Taketa, K., and Pogell, B. M. (1965) J. Biol. Chem. 240, 651-662[Free Full Text]
8. Nimmo, H. G., and Tipton, K. F. (1975) Eur. J. Biochem. 58, 567-574[Abstract]
9. Stone, S. R., and Fromm, H. J. (1980) Biochemistry 19, 620-625[Medline] [Order article via Infotrieve]
10. McGrane, M. M., El-Maghrabi, M. R., and Pilkus, S. J. (1983) J. Biol. Chem. 258, 10445-10454[Abstract/Free Full Text]
11. Liu, F., and Fromm, H. J. (1988) J. Biol. Chem. 263, 9122-9128[Abstract/Free Full Text]
12. Sola, M. M., Oliver, F. J., Salto, R., Gutierrez, M., and Vargas, A. M. (1993) Int. J. Biochem. 25, 1963-1968[Medline] [Order article via Infotrieve]
13. Zhang, R., Villeret, V., Lipscomb, W. N., and Fromm, H. J. (1996) Biochemistry 35, 3038-3043[CrossRef][Medline] [Order article via Infotrieve]
14. Nimmo, H. G., and Tipton, K. F. (1975) Eur. J. Biochem. 58, 575-585[Abstract]
15. Zhang, Y., Liang, J.-Y., Huang, S., and Lipscomb, W. N. (1994) J. Mol. Biol. 244, 609-624[CrossRef][Medline] [Order article via Infotrieve]
16. Zhang, Y., Liang, J., Huang, S., Ke, H., and Lipscomb, W. N. (1993) Biochemistry 32, 1844-1857[Medline] [Order article via Infotrieve]
17. Choe, J.-Y., Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1998) Biochemistry 37, 11441-11450[CrossRef][Medline] [Order article via Infotrieve]
18. Ke, H., Zhang, G. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5243-5247[Abstract]
19. Ke, H., Zhang, Y., Liang, J.-Y., and Lipscomb, W. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2989-2993[Abstract]
20. Choe, J.-Y., Fromm, H. J., and Honzatko, R. B. (2000) Biochemistry 39, 8565-8574[CrossRef][Medline] [Order article via Infotrieve]
21. Scheffler, J. E., and Fromm, H. J. (1986) Biochemistry 25, 6659-6665[Medline] [Order article via Infotrieve]
22. Liu, F., and Fromm, H. J. (1990) J. Biol. Chem. 265, 7401-7406[Abstract/Free Full Text]
23. Nelson, S. W., Kurbanov, F. T., Honzatko, R. B., and Fromm, H. J. (2001) J. Biol. Chem. 276, 6119-6124[Abstract/Free Full Text]
24. Kurbanov, F. T., Choe, J.-Y., Honzatko, R. B., and Fromm, H. J. (1998) J. Biol. Chem. 273, 17511-17516[Abstract/Free Full Text]
25. Nelson, S. W., Choe, J. Y., Honzatko, R. B., and Fromm, H. J. (2000) J. Biol. Chem. 275, 29986-29992[Abstract/Free Full Text]
26. Nelson, S. W., Iancu, C. V., Choe, J. Y., Honzatko, R. B., and Fromm, H. J. (2000) Biochemistry 39, 11100-11106[CrossRef][Medline] [Order article via Infotrieve]
27. Villeret, V., Huang, S., Fromm, H. J., and Lipscomb, W. N. (1995) Proc. Natl., Acad. Sci. U. S. A. 92, 8916-8920[Abstract]
28. Burton, V. A., Chen, M., Ong, W. C., Ling, T., Fromm, H. J., and Stayton, M. M. (1993) Biochem. Biophys. Res. Commun. 192, 511-517[CrossRef][Medline] [Order article via Infotrieve]
29. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
30. Bradford, M. M. (1976) Anal. Biochem. 72, 248-252[CrossRef][Medline] [Order article via Infotrieve]
31. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
32. CrystalClear (2001) Rigaku Molecular Structure Corporation, Orem, Utah
33. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, N., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crysallogr. Sect. D 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
34. McRee, D. E. (1992) J. Mol. Graphics 10, 44-46[CrossRef]
35. Engh, R. A., and Huber, R. (1991) Acta Crystallogr. Sect. A 47, 392-400[CrossRef]
36. Sheldrick, G. M., and Gould, R. O. (1995) Acta Crystallogr. Sect. B 51, 423-431[CrossRef]
37. Leatherbarrow, R. J. (1987) ENZFITTER: A Non-Linear Regression Data Analysis Program for the IBM PC , pp. 13-75, Elsevier Science Publishers B. V., Amsterdam.
38. Marcus, F., and Hosey, M. M. (1980) J. Biol. Chem. 255, 2481-2486[Free Full Text]
39. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524
40. Kraulis, J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
41. Pahler, A., Smith, J. L., and Hendrickson, W. A. (1990) Acta Crystallogr. Sect. A 46, 537-540[CrossRef][Medline] [Order article via Infotrieve]
42. Lee, A. G. (1971) The Chemistry of Thallium , Elsevier Publishing Company, New York
43. Kelley, N., Giroux, E. L., Lu, G., and Kantrowitz, E. R. (1996) Biochem. Biophys. Res. Commum. 219, 848-852[CrossRef][Medline] [Order article via Infotrieve]
44. Choe, J-Y., Iancu, C. V., Fromm, H. J., and Honzatko, R. B. (2003) J. Biol. Chem. 278, 16015-16020


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.