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• W2080434163 abstract "Fructose-1,6-bisphosphatase requires divalent cations (Mg2+, Mn2+, or Zn2+) for catalysis, but a diverse set of monovalent cations (K+, Tl+, Rb+, or NH4+) 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 aK a 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. Fructose-1,6-bisphosphatase requires divalent cations (Mg2+, Mn2+, or Zn2+) for catalysis, but a diverse set of monovalent cations (K+, Tl+, Rb+, or NH4+) 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 aK a 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. fructose-1,6-bisphosphatase fructose 6-phosphate, F16P2, fructose 1,6-bisphosphate fructose 2,6-bisphosphate protein data bank 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) (1Krebs H.A. Weber G. Advances in Enzyme Regulation. 1. Pergamon Press Ltd., London1963: 385-400Google Scholar, 2Marcus F. Veneziabe C.M. The Regulation of Carbohydrate Formation and Utilization in Mammals. University Park Press, Baltimore1981: 269-290Google Scholar, 3Benkovic S.J. de Maine M.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 45-82PubMed Google Scholar, 4Hers H.G. Hue L. Annu. Rev. Biochem. 1983; 52: 617-653Crossref PubMed Scopus (399) Google Scholar, 5Tejwani G.A. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 121-194PubMed Google Scholar, 6Pilkus S.J. El-Maghrabi M.R. Claus T.H. Annu. Rev. Biochem. 1988; 57: 755-783Crossref PubMed Scopus (317) Google Scholar). 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 (7Taketa K. Pogell B.M. J. Biol. Chem. 1965; 240: 651-662Abstract Full Text PDF PubMed Google Scholar, 8Nimmo H.G. Tipton K.F. Eur. J. Biochem. 1975; 58: 567-574Crossref PubMed Scopus (50) Google Scholar, 9Stone S.R. Fromm H.J. Biochemistry. 1980; 19: 620-625Crossref PubMed Scopus (30) Google Scholar). F26P2 competes with F16P2 for the active site (10McGrane M.M. El-Maghrabi M.R. Pilkus S.J. J. Biol. Chem. 1983; 258: 10445-10454Abstract Full Text PDF PubMed Google Scholar, 11Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar, 12Sola M.M. Oliver F.J. Salto R. Gutierrez M. Vargas A.M. Int. J. Biochem. 1993; 25: 1963-1968Crossref PubMed Scopus (11) Google Scholar). 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 (8Nimmo H.G. Tipton K.F. Eur. J. Biochem. 1975; 58: 567-574Crossref PubMed Scopus (50) Google Scholar, 14Nimmo H.G. Tipton K.F. Eur. J. Biochem. 1975; 58: 575-585Crossref PubMed Scopus (37) Google Scholar). The mammalian enzyme is a homotetramer and exists in distinct conformational states, depending on the relative concentrations of active site ligands and AMP (15Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (92) Google Scholar). With or without metal cofactors and/or other active site ligands, but in the absence of AMP, FBPase is in its R-state (16Zhang Y. Liang J. Huang S. Ke H. Lipscomb W.N. Biochemistry. 1993; 32: 1844-1857Crossref PubMed Scopus (96) Google Scholar, 17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar). In the presence of AMP, however, the top pair of subunits rotates 17° relative to the bottom pair, resulting in the T-state conformer (18Ke H. Zhang G.Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5243-5247Crossref PubMed Scopus (119) Google Scholar, 19Ke H. Zhang Y. Liang J.-Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2989-2993Crossref PubMed Scopus (57) Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar). The minimum distance separating AMP molecules from any given active site is ∼28 Å (18Ke H. Zhang G.Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5243-5247Crossref PubMed Scopus (119) Google Scholar, 19Ke H. Zhang Y. Liang J.-Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2989-2993Crossref PubMed Scopus (57) Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar). Yet studies in kinetics, NMR, fluorescence, and x-ray crystallography reveal competition between AMP and divalent cations (11Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar, 21Scheffler J.E. Fromm H.J. Biochemistry. 1986; 25: 6659-6665Crossref PubMed Scopus (32) Google Scholar, 22Liu F. Fromm H.J. J. Biol. Chem. 1990; 265: 7401-7406Abstract Full Text PDF PubMed Google Scholar). 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 (17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar, 23Nelson S.W. Kurbanov F.T. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2001; 276: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The engaged conformation is arguably required for the high affinity association of divalent cations and in stabilizing the transition state (17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar, 23Nelson S.W. Kurbanov F.T. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2001; 276: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 24Kurbanov F.T. Choe J.-Y. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1998; 273: 17511-17516Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25Nelson S.W. Choe J.Y. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2000; 275: 29986-29992Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 26Nelson S.W. Iancu C.V. Choe J.Y. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar). 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 (23Nelson S.W. Kurbanov F.T. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2001; 276: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In addition to the absolute requirement for divalent cations, certain monovalent cations (K+ and Tl+ among others) further enhance catalysis by FBPase (3Benkovic S.J. de Maine M.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 45-82PubMed Google Scholar, 5Tejwani G.A. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 121-194PubMed Google Scholar, 13Zhang R. Villeret V. Lipscomb W.N. Fromm H.J. Biochemistry. 1996; 35: 3038-3043Crossref PubMed Scopus (47) Google Scholar). 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 K a for Mg2+, and without exception also abolish K+-induced effects on catalysis (25Nelson S.W. Choe J.Y. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2000; 275: 29986-29992Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,26Nelson S.W. Iancu C.V. Choe J.Y. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar). On the other hand, K+ does not increase the fraction of subunits with an engaged loop in the presence of saturating Mg2+/F6P/Pi (26Nelson S.W. Iancu C.V. Choe J.Y. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar). Hence, improved catalysis comes from a more stable transition state in the presence of K+. Previous work of Lipscomb and co-workers (27Villeret V. Huang S. Fromm H.J. Lipscomb W.N. Proc. Natl., Acad. Sci. U. S. A. 1995; 92: 8916-8920Crossref PubMed Scopus (73) Google Scholar) 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 K a 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. 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 colistrain DF657 came from the Genetic Stock Center at Yale University. Expression and purification of FBPase followed the procedures of Burton et al. (28Burton V.A. Chen M. Ong W.C. Ling T. Fromm H.J. Stayton M.M. Biochem. Biophys. Res. Commun. 1993; 192: 511-517Crossref PubMed Scopus (20) Google Scholar) with minor modifications (20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar). 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 (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205463) Google Scholar) and by the Bradford assay (30Bradford M.M. Anal. Biochem. 1976; 72: 248-252Crossref PubMed Scopus (211739) Google Scholar), respectively. 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 mmKPi, 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 (17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar) and result in crystals that in some cases diffract to near atomic resolution (see below). 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 mmTl+ 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 CuKα radiation, passed through an Osmic confocal mirror system. Data from synchrotron sources were reduced and scaled by Denzo/Scalepack (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38231) Google Scholar). Data from the R-AXIS IV++ were processed with CrystalClear (32CrystalClear (2001) Rigaku Molecular Structure Corporation, Orem, Utah.Google Scholar). 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 either1CNQ or 1EYI, less ligands and water molecules. Electron density maps were calculated using CNS (33Brunger 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. Warren G.L. Acta Crysallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16909) Google Scholar). 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 (34McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar). Models were refined against x-ray data using CNS with force constants and parameters of stereochemistry from Engh and Huber (35Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2533) Google Scholar). Final cycles of refinement used SHELX (36Sheldrick G.M. Gould R.O. Acta Crystallogr. Sect. B. 1995; 51: 423-431Crossref PubMed Scopus (149) Google Scholar) 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 2F obs−F calc omit maps and anomalous difference maps. Assays employed the coupling enzymes, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (1Krebs H.A. Weber G. Advances in Enzyme Regulation. 1. Pergamon Press Ltd., London1963: 385-400Google Scholar). The coupling enzymes were dialyzed exhaustively in order to remove NH4+. 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 (23Nelson S.W. Kurbanov F.T. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2001; 276: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). 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 (37Leatherbarrow R.J. ENZFITTER: A Non-Linear Regression Data Analysis Program for the IBM PC. Elsevier Science Publishers B. V., Amsterdam.1987: 13-75Google Scholar). The Hill coefficient for Tl+ activation came from a least squares fit of the following shown in Equation 1, V=[VmSn/(Ka+Sn)]+3.0863Equation 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,K a 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. 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 (38Marcus F. Hosey M.M. J. Biol. Chem. 1980; 255: 2481-2486Abstract Full Text PDF PubMed Google Scholar) 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 aK a of 1.3 ± 0.1 mm. (TheK a 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% (26Nelson S.W. Iancu C.V. Choe J.Y. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar), with a Hill coefficient of unity and a K a of 17 mm (13Zhang R. Villeret V. Lipscomb W.N. Fromm H.J. Biochemistry. 1996; 35: 3038-3043Crossref PubMed Scopus (47) Google Scholar). Conditions of crystallization of R-state FBPase differ from those employed in past work (17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar) 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 (44Choe J-Y. Iancu C.V. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2003; 278: 16015-16020Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), 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 (17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar, 20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar). The side chain of Tyr57 in the engaged conformation occupies a hydrophobic pocket (26Nelson S.W. Iancu C.V. Choe J.Y. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar). 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 (20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar). As these complexes have been reported in detail in prior publications (17Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (60) Google Scholar,20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar), we focus here on changes in the active site in response to different conditions of crystal growth.Table IStatistics of data collection and refinementT1+ concentration (mm)01-aEDTA absent.11-aEDTA absent.51-aEDTA absent.201-aEDTA absent.1001-aEDTA absent.11-bEDTA present at 0.2 mm.51-bEDTA present at 0.2 mm.201-bEDTA present at 0.2 mm.201-cEDTA absent and AMP present at 5 mm.Resolution limit (Å)1.91.81.92.121.91.92.152.15Wavelength of x-rays1.541780.97951.54150.97950.97951.54151.54151.54150.9795Space groupI222I222I222I222I222I222I222I222P21212No. of measurements1701562388352881389286997976182349172612163129293146No. of unique refl.260983031128039176042071926022271061954540229Completeness of data (%): Overall89.287.495.986.582.88992.999.999.9 Last shell49.286.869.943.749.248.861.399.199.9R sym1-dR sym = Σ j Σ i ‖1 ij − 〈1 j 〉‖/Σ i Σ j I ij, where iruns over multiple observations of the same intensity, and jruns over all crystallographically unique intensities.0.0380.0750.0230.030.0520.0240.0210.0710.104No. of reflections in refinement1-eAll data in the resolution ranges indicated.243512898326216156521834124246253431855238218No. of atoms1-fIncludes hydrogens linked to polar atoms.272328152737278726892743276527105519No. of solvent sites183253176211146199197165425R-factor1-gR-factor = Σ‖‖F obs‖−‖F calc‖‖/Σ‖F obs‖, ‖F obs‖ > 0.0.19270.20010.19680.17030.20420.18430.19150.21080.1824R free1-hR-factor based upon 5% of the data randomly culled and not used in the refinement.0.24610.24710.25590.24870.26610.23660.24530.28010.2511Mean B (Å2): Entire protein28.224.625.828.228.624.825.832.624.4 Residues 61–69515061968251699490Root mean square deviations: Bond lengths (Å)0.0070.0080.0070.0060.0060.0070.0070.0060.006 Bond angles (degree)1.91.91.81.71.81.81.91.81.8 Dihedral angles (degree)25.624.824.624.925.225.12525.325.7 Improper angles (degree)1.331.271.251.281.231.241.271.271.71Occupancies (%) of metal sites: Site 1, Mg2+/T1+100/084/1095/658/30/080/1075/578/2100/0 Site 2, Mg2+/T11100/0100/068/550/7100/080/382/460/80/15 Site 3, Mg2+501000040000 Site 3a, T1+001130330133015 Site 3b, T1+001018184101710 Site 4, T1+0092533061415 Site 5, T1+00102535092314Occupancies (%) of the 1-OH group of F6P: Productive100503500504000 Nonproductive050651001005060100100Mg2+ is present in all complexes at 5 mm.1-a EDTA absent.1-b EDTA present at 0.2 mm.1-c EDTA absent and AMP present at 5 mm.1-d R sym = Σ j Σ i ‖1 ij − 〈1 j 〉‖/Σ i Σ j I ij, where iruns over multiple observations of the same intensity, and jruns over all crystallographically unique intensities.1-e All data in the resolution ranges indicated.1-f Includes hydrogens linked to polar atoms.1-g R-factor = Σ‖‖F obs‖−‖F calc‖‖/Σ‖F obs‖, ‖F obs‖ > 0.1-h R-factor based upon 5% of the data randomly culled and not used in the refinement. Open table in a new tab 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 (λ = 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 (λ = 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 (41Pahler A. Smith J.L. Hendrickson W.A. Acta Crystallogr. Sect. A. 1990; 46: 537-540Crossref PubMed Scopus (53) Google Scholar). (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 (27Villeret V. Huang S. Fromm H.J. Lipscomb W.N. Proc. Natl., Acad. Sci. U. S. A. 1995; 92: 8916-8920Crossref PubMed Scopus (73) Google Scholar). 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 (20Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (62) Google Scholar). The Mg2+ at site 3 and its coordinated water molecule are probably important to the catalyti" @default.
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• W2080434163 cites W1990292401 @default.
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• W2080434163 cites W1992738264 @default.
• W2080434163 cites W1992992784 @default.
• W2080434163 cites W1995017064 @default.
• W2080434163 cites W2003809510 @default.
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• W2080434163 cites W2024729288 @default.
• W2080434163 cites W2028231353 @default.
• W2080434163 cites W2028697349 @default.
• W2080434163 cites W2040300526 @default.
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• W2080434163 cites W2046954314 @default.
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• W2080434163 cites W2060503091 @default.
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• W2080434163 cites W2088669134 @default.
• W2080434163 cites W2089543391 @default.
• W2080434163 cites W2100837269 @default.
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• W2080434163 cites W2116275717 @default.
• W2080434163 cites W2128421575 @default.
• W2080434163 cites W2134883783 @default.
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• W2080434163 cites W2165992714 @default.
• W2080434163 cites W2398388055 @default.
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