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Structure of Inhibited Fructose-1,6-bisphosphatase from Escherichia coli

2007; Elsevier BV; Volume: 282; Issue: 34 Linguagem: Inglês

10.1074/jbc.m703580200

1083-351X

Justin K. Hines, Claire E. Kruesel, Herbert J. Fromm, Richard B. Honzatko,

Biochemical and Molecular Research

Allosteric activation of fructose-1,6-bisphosphatase (FBPase) from Escherichia coli by phosphoenolpyruvate implies rapid feed-forward activation of gluconeogenesis in heterotrophic bacteria. But how do such bacteria rapidly down-regulate an activated FBPase in order to avoid futile cycling? Demonstrated here is the allosteric inhibition of E. coli FBPase by glucose 6-phosphate (Glc-6-P), the first metabolite produced upon glucose transport into the cell. FBPase undergoes a quaternary transition from the canonical R-state to a T-like state in response to Glc-6-P and AMP ligation. By displacing Phe15, AMP binds to an allosteric site comparable with that of mammalian FBPase. Relative movements in helices H1 and H2 perturb allosteric activator sites for phosphoenolpyruvate. Glc-6-P binds to allosteric sites heretofore not observed in previous structures, perturbing subunits that in pairs form complete active sites of FBPase. Glc-6-P and AMP are synergistic inhibitors of E. coli FBPase, placing AMP/Glc-6-P inhibition in bacteria as a possible evolutionary predecessor to AMP/fructose 2,6-bisphosphate inhibition in mammalian FBPases. With no exceptions, signature residues of allosteric activation appear in bacterial sequences along with key residues of the Glc-6-P site. FBPases in such organisms may be components of metabolic switches that allow rapid changeover between gluconeogenesis and glycolysis in response to nutrient availability. Allosteric activation of fructose-1,6-bisphosphatase (FBPase) from Escherichia coli by phosphoenolpyruvate implies rapid feed-forward activation of gluconeogenesis in heterotrophic bacteria. But how do such bacteria rapidly down-regulate an activated FBPase in order to avoid futile cycling? Demonstrated here is the allosteric inhibition of E. coli FBPase by glucose 6-phosphate (Glc-6-P), the first metabolite produced upon glucose transport into the cell. FBPase undergoes a quaternary transition from the canonical R-state to a T-like state in response to Glc-6-P and AMP ligation. By displacing Phe15, AMP binds to an allosteric site comparable with that of mammalian FBPase. Relative movements in helices H1 and H2 perturb allosteric activator sites for phosphoenolpyruvate. Glc-6-P binds to allosteric sites heretofore not observed in previous structures, perturbing subunits that in pairs form complete active sites of FBPase. Glc-6-P and AMP are synergistic inhibitors of E. coli FBPase, placing AMP/Glc-6-P inhibition in bacteria as a possible evolutionary predecessor to AMP/fructose 2,6-bisphosphate inhibition in mammalian FBPases. With no exceptions, signature residues of allosteric activation appear in bacterial sequences along with key residues of the Glc-6-P site. FBPases in such organisms may be components of metabolic switches that allow rapid changeover between gluconeogenesis and glycolysis in response to nutrient availability. Fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase; EC 3.1.3.11; FBPase) 3The abbreviations used are: FBPasefructose-1,6-bisphosphataseFru-6-Pfructose 6-phosphateFru-16-P2, fructose 1,6-bisphosphateFru-2, 6-P2fructose 2,6-bisphosphatePFKfructose-6-phosphate-1-kinasePEPphosphoenolpyruvateGlc-6-Pglucose 6-phosphateAnG6P1,5-anhydro-d-glucitol 6-phosphateMES2-(N-morpholino)ethanesulfonic acid 3The abbreviations used are: FBPasefructose-1,6-bisphosphataseFru-6-Pfructose 6-phosphateFru-16-P2, fructose 1,6-bisphosphateFru-2, 6-P2fructose 2,6-bisphosphatePFKfructose-6-phosphate-1-kinasePEPphosphoenolpyruvateGlc-6-Pglucose 6-phosphateAnG6P1,5-anhydro-d-glucitol 6-phosphateMES2-(N-morpholino)ethanesulfonic acid catalyzes the hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and Pi and is a principal regulatory enzyme in gluconeogenesis (1Benkovic S.T. de Maine M.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 45-82PubMed Google Scholar, 2Tejwani G.A. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 121-194PubMed Google Scholar). Primary sequence comparisons infer five nonhomologous FBPases in living organisms. All five types exist in various prokaryotes, but the Type I enzyme is the only form in eukaryotes (3Fraenkel D.G. Horecker B.L. J. Bacteriol. 1965; 90: 837-842Crossref PubMed Google Scholar, 4Fraenkel D.G. Pontremoli S. Horecker B.L. Arch. Biochem. Biophys. 1966; 114: 4-12Crossref PubMed Scopus (36) Google Scholar, 5Sato T. Imanaka H. Rashid N. Fukui T. Atomi H. Imanaka T. J Bacteriol. 2004; 186: 5799-5807Crossref PubMed Scopus (81) Google Scholar, 6Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol. 2000; 182: 5624-5627Crossref PubMed Scopus (87) Google Scholar, 7Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2006; 281: 18386-18393Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The enteric bacterium Escherichia coli requires Type I FBPase for growth on gluconeogenic substrates (3Fraenkel D.G. Horecker B.L. J. Bacteriol. 1965; 90: 837-842Crossref PubMed Google Scholar). That enzyme is subject to regulation by metabolites (4Fraenkel D.G. Pontremoli S. Horecker B.L. Arch. Biochem. Biophys. 1966; 114: 4-12Crossref PubMed Scopus (36) Google Scholar, 7Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2006; 281: 18386-18393Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 9Babul J. Guixe V. Arch. Biochem. Biophys. 1983; 225: 944-949Crossref PubMed Scopus (37) Google Scholar). fructose-1,6-bisphosphatase fructose 6-phosphate 6-P2, fructose 1,6-bisphosphate fructose 2,6-bisphosphate fructose-6-phosphate-1-kinase phosphoenolpyruvate glucose 6-phosphate 1,5-anhydro-d-glucitol 6-phosphate 2-(N-morpholino)ethanesulfonic acid fructose-1,6-bisphosphatase fructose 6-phosphate 6-P2, fructose 1,6-bisphosphate fructose 2,6-bisphosphate fructose-6-phosphate-1-kinase phosphoenolpyruvate glucose 6-phosphate 1,5-anhydro-d-glucitol 6-phosphate 2-(N-morpholino)ethanesulfonic acid Type I FBPases are tetramers with subunits labeled C1–C4 by convention. The porcine enzyme, the most studied of all mammalian FBPases, adopts distinct quaternary states called R, IT, and T (10Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar, 11Ke H. Zhang Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5243-5247Crossref PubMed Scopus (119) Google Scholar, 12Shyur L.F. Aleshin A.E. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1996; 271: 33301-33307Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Iancu C.V. Mukund S. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2005; 280: 19737-19745Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 14Choe J.-Y. Nelson S.W. Arienti K.L. Axe F.U. Collins T.L. Jones T.K. Kimmich R.D. Newman M.J. Norvell K. Ripka W.C. Romano S.J. Short K.M. Slee D.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2003; 278: 51176-51183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The porcine liver enzyme takes on an active R-state conformation in the absence of the inhibitor AMP, whereas the AMP-bound form assumes an inactive T-state that differs from the R-state by a 15° rotation between “top” and “bottom” subunit pairs (10Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar, 11Ke H. Zhang Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5243-5247Crossref PubMed Scopus (119) Google Scholar, 12Shyur L.F. Aleshin A.E. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1996; 271: 33301-33307Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Iancu C.V. Mukund S. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2005; 280: 19737-19745Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The competitive inhibitor fructose 2,6-bisphosphate (Fru-2,6-P2) converts the enzyme to an intermediate state (IT), which lies 12–13° away from the R-state (14Choe J.-Y. Nelson S.W. Arienti K.L. Axe F.U. Collins T.L. Jones T.K. Kimmich R.D. Newman M.J. Norvell K. Ripka W.C. Romano S.J. Short K.M. Slee D.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2003; 278: 51176-51183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The constitutive expression of both fructose-6-phosphate-1-kinase (PFK) and FBPase in bacteria demands a strategy of coordinate metabolite regulation (3Fraenkel D.G. Horecker B.L. J. Bacteriol. 1965; 90: 837-842Crossref PubMed Google Scholar, 8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 15Pilkis S.J. El-Maghrabi M.R. Claus T.H. Annu. Rev. Biochem. 1988; 57: 755-783Crossref PubMed Scopus (322) Google Scholar). Expression levels of FBPase and PFK in E. coli vary only 2–3-fold between glycolytic and gluconeogenic conditions, yet futile cycling remains low under both conditions (16Chin A.M. Feldheim D.A. Saier Jr., M.H. J. Bacteriol. 1989; 171: 2424-2434Crossref PubMed Scopus (64) Google Scholar, 17Oh M.-K. Rohlin L. Kao K.C. Liao J.C. J. Biol. Chem. 2002; 277: 13175-13183Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 18Chambost J.-P. Fraenkel D.G. J. Biol. Chem. 1980; 255: 2867-2869Abstract Full Text PDF PubMed Google Scholar, 19Daldal F. Fraenkel D.G. J. Bacteriol. 1983; 153: 390-394Crossref PubMed Google Scholar). Phosphoenolpyruvate (PEP) and citrate are probable in vivo allosteric effectors in the feed-forward activation of E. coli FBPase and gluconeogenesis (8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Activators of E. coli FBPase bind to allosteric sites between subunit pairs C1–C2 and C3–C4, stabilizing a quaternary state nearly identical to the R-state of the mammalian enzyme (8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Moreover, concentrations of PEP rise coordinately with the availability of nonglucose nutrients (20Franzen J.S. Binkley S.B. J. Biol. Chem. 1960; 236: 515-519Abstract Full Text PDF Google Scholar, 21Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar). But then how does E. coli respond to a sudden influx of glucose? Eukaryotic organisms produce Fru-2,6-P2 to activate PFK and inhibit FBPase. Saccharomyces cerevisiae, for instance, produces inhibitory levels of Fru-2,6-P2 within 1 min of glucose exposure (22Lederer B. Vissers S. Van Schaftingen E. Hers H.-G. Biochem. Biophys. Res. Commun. 1981; 103: 1281-1287Crossref PubMed Scopus (52) Google Scholar). Heterotrophic bacteria, such as E. coli, however, have no known mechanism for the rapid inhibition of FBPase in response to a sudden influx of glucose. Glucose is immediately phosphorylated to glucose 6-phosphate (Glc-6-P) upon transport into the bacterial cell via the action of either the PEP phosphotransferase system or glucokinase (23Fraenkel D.G. Neidhardt F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 189-198Google Scholar). Glc-6-P levels vary as much as 10-fold between E. coli grown on different carbon sources and can rapidly fluctuate 5–7-fold in response to nutrient availability (21Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar). Glc-6-P, being downstream of the FBPase reaction and subject to nutrient-induced variations in concentration, has the desired attributes of a dynamic regulator of FBPase. A mutant strain of E. coli, lacking phosphoglucose isomerase and glucose-6-phosphate dehydrogenase activities, accumulates large amounts of intracellular Glc-6-P (∼50 mm, normal range 0.2–2 mm); its failure to grow on glycerol suggests in vivo inhibition of gluconeogenesis by Glc-6-P (21Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar, 24Fraenkel D.G. J. Biol. Chem. 1968; 243: 6451-6457Abstract Full Text PDF PubMed Google Scholar). Fifty percent inhibition of impure FBPase, however, required 10 mm Glc-6-P, undermining a physiological role for Glc-6-P in the regulation of FBPase (24Fraenkel D.G. J. Biol. Chem. 1968; 243: 6451-6457Abstract Full Text PDF PubMed Google Scholar). We demonstrate here the inhibition of purified E. coli FBPase (specific activity 1000-fold higher than that used in Ref. 24Fraenkel D.G. J. Biol. Chem. 1968; 243: 6451-6457Abstract Full Text PDF PubMed Google Scholar) at physiologically relevant concentrations of Glc-6-P. Moreover, Glc-6-P enhances AMP inhibition by as much as 10-fold, even in the presence of 1 mm PEP. The latter property might allow Glc-6-P to override PEP activation of gluconeogenesis. Glc-6-P binds to distinct allosteric sites never before observed in FBPase structures, shearing subunits that pair off in forming complete active sites. E. coli FBPase, ligated by Glc-6-P and AMP, is in a T-like state, similar to the IT-state of the porcine enzyme. AMP displaces the side chain of Phe15 and axially dislocates helices H1 and H2 in opposite directions. Helix displacement distorts the PEP activation site, destabilizes the R-state, and allows Arg80 to stabilize the T-like state through its interaction with Glu6 from a symmetry-related subunit. Arg80 and Glu6 are conserved in organisms possessing signature residues of the PEP and Glc-6-P sites. Similarities between Glc-6-P and Fru-2,6-P2 inhibition suggest an evolutionary linkage; both inhibitors are one-reaction products of Fru-6-P, and both inhibit their respective FBPases synergistically with AMP. Materials—Fru-1,6-P2, Glc-6-P, Fru-6-P, AMP, NADP+, and ammonium molybdate came from Sigma; PEP was from MP Biomedicals; and zinc acetate, ascorbic acid, and sulfuric acid were from Fisher. 1,5-Anhydro-d-glucitol 6-phosphate (AnG6P) was synthesized from 1,5-anhydro-d-glucitol (Toronto Research Chemicals) and quantified as previously described (25Ferrari R.A. Mandelstam P. Crane R.K. Arch. Biochem. Biophys. 1959; 80: 372-377Crossref Scopus (27) Google Scholar, 26Drueckes P. Schinzel R. Palm D. Anal. Biochem. 1995; 230: 173-177Crossref PubMed Scopus (86) Google Scholar). Glucose-6-phosphate dehydrogenase, phosphoglucose isomerase, and alkaline phosphatase were from Roche Applied Science. All other chemicals were of reagent grade. Enzyme Isolation and Purity—Native and selenomethionine-substituted E. coli FBPases, prepared as previously described (7Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2006; 281: 18386-18393Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), are structurally and kinetically indistinguishable. Preparations of selenomethionine-substituted enzyme were used for structure determinations and native enzyme for kinetics. Purified enzymes migrate as single bands on SDS-PAGE, have specific activities of 35–40 units/mg, and have a single residue type (methionine or selenomethionine) at the N terminus. Kinetic Experiments—Assays of E. coli FBPase use coupling enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase and monitor the formation of NADPH at 22 °C by either fluorescence emission at 470 nm or absorbance at 340 nm (13Iancu C.V. Mukund S. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2005; 280: 19737-19745Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Assay mixtures (total volume, 2 ml) included 50 mm Hepes, pH 7.5, 100 μm EDTA, and 150 μm NADP+ with up to saturating levels of Fru-1,6-P2 (40 μm) and MgCl2 (10 mm). Assays were initiated either by the addition of 1.4 μg of enzyme (enzyme-initiated assays) or by incubating the enzyme in assay mixtures for 1 h at 22 °C without MgCl2 and then initiating the reaction by the addition of metal (enzyme incubation assays). Glc-6-P is an intermediate in the coupled assay. Hence, studies of Glc-6-P inhibition monitor FBPase turnover by the evolution of Pi (26Drueckes P. Schinzel R. Palm D. Anal. Biochem. 1995; 230: 173-177Crossref PubMed Scopus (86) Google Scholar). Reducing reagent was prepared daily (26Drueckes P. Schinzel R. Palm D. Anal. Biochem. 1995; 230: 173-177Crossref PubMed Scopus (86) Google Scholar) with the addition of H2SO4 to a final concentration of 1 n (4Fraenkel D.G. Pontremoli S. Horecker B.L. Arch. Biochem. Biophys. 1966; 114: 4-12Crossref PubMed Scopus (36) Google Scholar). Assay mixtures (50 mm Hepes, pH 7.5, 0.1 mm EDTA, 0.5 mm Fru-1,6-P2, 0.75 μg of enzyme, and varying amounts of PEP, AMP, and Glc-6-P in a total volume of 60 μl) were incubated in 96-well microtiter plates (Evergreen) at 25 °C for 1 h prior to the initiation of the reaction by the addition of 5 mm MgCl2. The addition of 250 μl of reducing agent at fixed intervals quenched reactions. Plates were sealed and incubated at 25 °C for at least 2 h to allow color development. Absorbances (λ = 655 nm) were measured on a Bio-Rad Benchmark microplate reader. Blank-corrected standard curves relating A655 to [Pi] (0–1.5 mm) were linear (R2 > 0.998). Determinations of kinetic mechanism require low concentrations of substrate and result in levels of Pi undetectable by the phosphate release assay. Such determinations employed the coupled assay with AnG6P as the inhibitor. AnG6P does not interfere with enzymes of the coupled assay (25Ferrari R.A. Mandelstam P. Crane R.K. Arch. Biochem. Biophys. 1959; 80: 372-377Crossref Scopus (27) Google Scholar). Initial rate data taken at saturating substrate, fixed effector, and systematically varied inhibitor concentrations were fit to a Hill equation, V=V∞+(Vmax−V∞)/((I/I0.5)n+1)Eq. 1 where I represents the inhibitor concentration; V, Vmax, and V∞ are the velocity, maximum velocity (at I = 0), and the limiting velocity (at I saturating); n is the Hill coefficient associated with the inhibitor; and I0.5 is the inhibitor concentration at 50% inhibition. Data taken from experiments in which inhibitor/substrate or inhibitor/metal concentrations were varied systematically were fit to rapid equilibrium models for competitive, uncompetitive, noncompetitive, and mixed inhibition, fixing the Hill coefficient of the ligand (n = 1or n = 2) as circumstances dictate. AMP behaves cooperatively (n = 2) in enzyme-initiated assays or in the presence of PEP and noncooperatively (n = 1) in enzyme incubation assays in the absence of PEP (8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Kinetic data were fit to models using the programs IGORPRO (WaveMetrics) or DYNAFIT (27Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1354) Google Scholar). Estimates of in vivo metabolite concentrations in E. coli were determined as previously described (8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Crystallization of the AMP·Glc-6-P Complex—Crystals were grown by hanging drop in vapor diffusion VDX plates (Hampton Research). Solutions of sucrose and polyethylene glycol had 0.05% (w/v) NaN3 to reduce microbial growth. All other solutions were sterile filtered prior to use. AMP/Glc-6-P-bound FBPase crystals grew from droplets containing 2 μl of a protein solution (15 mg/ml enzyme, 20 mm dithiothreitol, 0.1 mm EDTA, 5 mm Fru-1,6-P2, 5 mm MgCl2, and 5 mm AMP) and 2 μlof a precipitant solution (50 mm MES-NaOH, pH 6.5, 13% (w/v) polyethylene glycol 10,000, and 20% (w/v) sucrose) and were equilibrated over 500 μl of precipitant solution. All components of the protein solution were combined, except AMP, and incubated at room temperature for 30 min, allowing for the enzyme-catalyzed equilibration of Fru-1,6-P2, Fru-6-P, and Glc-6-P prior to the addition of AMP. (FBPase isolated as described here has endogenous phosphoglucose isomerase activity that produces Glc-6-P from Fru-6-P. Purifying a C-terminal polyhistidyl-tagged FBPase on nickel-nitrilotriacetic acid-agarose (Novagen) removes the trace impurity, but the tagged FBPase has undetermined properties of crystallization). 4J. K. Hines and R. B. Honzatko, unpublished observations. Rodlike crystals (0.2 × 0.2 × 0.8 mm) grew within 3 days at 22 °C. Crystals were cryoprotected by immersion for 60 s in a solution of 18% (w/v) polyethylene glycol 20,000, 20% (v/v) 2-methyl-2,4-pentanediol, and 50 mm MES-NaOH, pH 6.5, supplemented with dithiothreitol, EDTA, and ligands immediately prior to freezing in a cold stream of nitrogen. Data Collection—Crystals were screened at Iowa State University on a Rigaku R-AXIS IV++ rotating anode/image plate system using CuKα radiation from an Osmic confocal optics system at a temperature of 110 K. Data were collected from a single crystal at 100 K on Beamline 4.2.2 of the Advanced Light Source, Lawrence Berkeley Laboratory. The program d*trek (28Pflugrath J.W. Acta Crystallogr. Sect. D. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar) was used to index, integrate, scale, and merge intensities, which were then converted to structure factors using the CCP4 (29Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) program TRUNCATE (30French G.S. Wilson K.S. Acta Crystallogr. Sect. A. 1978; 34: 517-525Crossref Scopus (892) Google Scholar). Structure Determination and Refinement—The program AMORE (31Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar) and a single subunit (less ligands) from the sulfate-bound E. coli FBPase structure (Protein Data Bank accession code 2GQ1) were used in constructing the asymmetric unit. Manual adjustments in the conformation of specific residues employed the program XTALVIEW (32McRee D.E. J. Mol. Graph. 1992; 10: 44-46Crossref Google Scholar). The initial model underwent simulated annealing from 3000 to 300 K in steps of 25 K, followed by 100 cycles of energy minimization and thermal parameter refinement using CNS (33Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). Force constants and parameters of stereochemistry were from Engh and Huber (34Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2543) Google Scholar). Restraints for thermal parameter refinement were as follows: 1.5 Å2 for bonded main-chain atoms, 2.0 Å2 for angle main-chain atoms and angle side-chain atoms, and 2.5 Å2 for angle side-chain atoms. Ligands (Fru-1,6-P2, Glc-6-P, AMP, Mg2+, HPO2–4, and Cl–) and water molecules were fit to omit electron density until no improvement in Rfree was evident. Water molecules with thermal parameters above 60 Å2 or more than 3.2 Å from the nearest hydrogen bonding partner were removed from the final model. Structure and Sequence Comparisons of FBPases—Tetramer models of E. coli and porcine FBPases were constructed from crystallographic asymmetric units and used in pairwise superpositions using the CCP4 programs PDBSET (35Evans P. PDBSET. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK1992Google Scholar) and LSQKAB (36Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2345) Google Scholar). Displacements between Cα positions were measured using XTALVIEW (32McRee D.E. J. Mol. Graph. 1992; 10: 44-46Crossref Google Scholar). The canonical R- and T-states of porcine FBPase have Protein Data Bank identifiers 1CNQ and 1EYK, respectively, whereas the IT- and IR-states have identifiers 1Q9D and 1YYZ. The measured angle of rotation between subunit pairs is sensitive to the set of residues used in the superposition. Hence, previously established residues employed in the comparison of E. coli and porcine FBPases were used for alignments (7Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2006; 281: 18386-18393Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Multiple sequence alignments of Type I FBPases employed the program ClustalW (37Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55635) Google Scholar). Inhibition of E. coli FBPase by Glc-6-P and AMP—AMP is a noncompetitive inhibitor of E. coli FBPase with respect to the substrate Fru-1,6-P2 under all methods of assay and in the presence or absence of PEP. Ki values range from 2 to 100μm, being sensitive to the conditions of assay and PEP concentration. At saturating concentrations of Fru-1,6-P2 (40 μm) and in the presence of 1 mm PEP, AMP inhibition is mixed with respect to Mg2+. The constant of dissociation for AMP from the enzyme·Fru-1,6-P2 complex (Ki = 13 ± 3μm) is 7-fold less than that for the dissociation of AMP from the enzyme·Fru-1,6-P2·Mg2+ complex (Kis = 94 ± 10 μm). (AMP is a competitive inhibitor with respect to Mg2+ for porcine FBPase (38Liu F. Fromm H.J. J. Biol. Chem. 1990; 265: 7401-7406Abstract Full Text PDF PubMed Google Scholar).) The determination of the kinetic mechanism of Glc-6-P inhibition employed the coupled assay and the analog AnG6P (which inhibits with 5–10-fold lower affinity than Glc-6-P). In assays with Fru-1,6-P2 or Mg2+ at saturating concentrations and 1 mm PEP, AnG6P inhibited E. coli FBPase noncompetitively with respect to both Fru-1,6-P2 (Ki = Kis = 660 ± 70 μm) and Mg2+ (Ki = Kis = 850 ± 70 μm). Glc-6-P inhibition, as measured by the phosphate release assay, varied with fixed concentrations of PEP and AMP (Fig. 1 and Table 1). PEP antagonizes AMP and Glc-6-P inhibition. I0.5 values for Glc-6-P and AMP inhibition increase 10- and 4-fold, respectively, over a range of 0–1 mm PEP. 500 μm Glc-6-P enhances AMP inhibition by 10-fold.TABLE 1I0.5 values for the inhibition of E. coli FBPase by AMP and Glc-6-P[PEP]I0.5 AMPI0.5 AMP with 500 μm Glc-6-PI0.5 Glc-6-Pμmμmμmμm08 ± 26 ± 138 ± 65012 ± 24 ± 1100 ± 20100031 ± 73 ± 2340 ± 40 Open table in a new tab Structure of the AMP·Glc-6-P Complex of E. coli FBPase (Protein Data Bank Accession Code 2Q8M)—Crystals belong to the space group P4122 (a = b = 124.6, c = 132.3 Å) with a C1–C4 dimer pair in the asymmetric unit (Fig. 2 and Table 2). Additional subunits related by crystallographic symmetry complete the biological tetramer. Four AMP and two Glc-6-P molecules bind in full occupancy to the tetramer. AMP molecules are at allosteric sites corresponding to those of the porcine tetramer (10Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar), whereas Glc-6-P molecules bind at allosteric sites that incorporate a 2-fold axis of molecular/crystallographic symmetry. Hence, Glc-6-P in the model adopts mutually exclusive modes of binding related by symmetry (Fig. 2). Chloride ions (a total of six per tetramer) and water molecules occupy conformationally altered sites associated with PEP activation in the R-state (8Hines J.H. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2007; 282: 11696-11704Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Fru-1,6-P2 and two Mg2+ are in each active site. The electron density for the 1-phosphoryl groups and Mg2+, however, is less robust than that for the rest of the ligand. A mixture of Fru-1,6-P2 and Fru-6-P probably binds to the active site. To reflect this circumstance, occupancy factors for 1-phosphoryl groups and Mg2+ are set to 0.5. Magnesium ions occupy loci analogous to metal sites 1 and 2 of porcine FBPase (39Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (59) Google Scholar) and have five and six coordinating oxygen atoms, respectively. A complete list of protein ligand interactions appears in Table 3. The model does not include residues 46–67 (most of the dynamic loop and the N-terminal end of helix H3) due to the absence of interpretable electron density; however, residues 42–46 at the N-terminal end of the loop occupy a position consistent with the disengaged conformation of the dynamic loop in T-state models of porcine FBPase (40Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar).TABLE 2Statistics of data collection and refinement of the AMP·Glc-6-P complexGlc-6-P·AMP E. coli FBPaseResolution (Å)37.8-2.05 (2.12-2.05)Total reflections/unique reflections154,407/63,536Average redundancy2.4 (2.5)Completeness (%)96.5 (95.7)RmergeaRmerge = ∑j∑i|Iij — 〈Ij〉|/∑i∑jIij, where i runs over multiple observations of the same intensity and j runs over all of the crystallographically unique intensities.0.068 (0.255)I/σ(I)9.2 (3.5)No. of atoms5305No. of solvent sites312RfactorbRfactor = ∑∥Fo| — |Fc∥/∑|Fo|, where |Fo| > 0.20.9RfreecRfree is the Rfactor based upon 10% of the data randomly culled and not used in the refinement.23.5Mean thermal parameters (Å2)Protein29Fru-1,6-P223Mg2+30AMP25Glc-6-P27Cl-57Root mean square deviationsBond lengths (Å)0.006Bond angles (degrees)1.2Dihedral angles (degrees)22.8Improper angles (degrees)0.7a Rmerge = ∑j∑i|Iij — 〈Ij〉|/∑i∑jIij, where i runs over multiple observations of the same intensity and j runs over all of the crystallographically unique intensities.b Rfactor = ∑∥Fo| — |Fc∥/∑|Fo|, where |Fo| > 0.c Rfree is the Rfactor based upon 10% of the data randomly culled and not used in the refinement.