A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance. A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance. Fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) 1The abbreviations used are: FBPasefructose-1,6-bisphosphataseF16P2fructose 1,6-bisphosphateF6Pfructose 6-phosphateF26P2fructose 2,6-bisphosphate.1The abbreviations used are: FBPasefructose-1,6-bisphosphataseF16P2fructose 1,6-bisphosphateF6Pfructose 6-phosphateF26P2fructose 2,6-bisphosphate. catalyzes a tightly regulated step of gluconeogenesis, the hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose 6-phosphate (F6P) and Pi (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). AMP and F26P2 (binding to allosteric and active sites, respectively) inhibit FBPase, while simultaneously activating its counterpart in glycolysis, fructose-6-phosphate 1-kinase (3Van Schaftingen E. Adv. Enzymol. Relat. Areas Mol. Biol. 1987; 59: 45-82Google Scholar, 4Pilkis S.J. El-Maghrabi M.R. Claus T.H. Annu. Rev. Biochem. 1988; 57: 755-783Crossref PubMed Scopus (317) Google Scholar). Biosynthesis and degradation of F26P2 is subject to hormonal control principally by glucagon and insulin (4Pilkis S.J. El-Maghrabi M.R. Claus T.H. Annu. Rev. Biochem. 1988; 57: 755-783Crossref PubMed Scopus (317) Google Scholar, 5Okar D.A. Lange A.J. Biofactors. 1999; 10: 1-14Crossref PubMed Scopus (119) Google Scholar). F26P2 enhances the binding of AMP to FBPase by up to an order of magnitude (6Pilkis S.J. El-Maghrabi R.M. McGrane M.M. Pilkis J. Claus T.H. J. Biol. Chem. 1981; 256: 3619-3622Abstract Full Text PDF PubMed Google Scholar). Hence, although intracellular concentrations of AMP remain relatively constant, AMP becomes a more potent inhibitor of FBPase as concentrations of F26P2 increase. AMP binds 28 Å away from the nearest active site and perhaps not surprisingly inhibits catalysis noncompetitively with respect to F16P2. Yet AMP is a competitive inhibitor of catalysis with respect to essential divalent cations (Mg2+, Mn2+, or Zn2+), all of which are in proximity to (and probably coordinate with) the 1-phosphoryl group of F16P2 (7Nimmo H.G. Tipton K.F. Eur. J. Biochem. 1975; 58: 567-574Crossref PubMed Scopus (50) Google Scholar, 8Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar, 9Scheffler J.E. Fromm H.J. Biochemistry. 1986; 25: 6659-6665Crossref PubMed Scopus (32) Google Scholar, 10Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar). fructose-1,6-bisphosphatase fructose 1,6-bisphosphate fructose 6-phosphate fructose 2,6-bisphosphate. fructose-1,6-bisphosphatase fructose 1,6-bisphosphate fructose 6-phosphate fructose 2,6-bisphosphate. FBPase is a homotetramer (subunit Mr of 37,000 (11Stone S.R. Fromm H.J. Biochemistry. 1980; 19: 620-625Crossref PubMed Scopus (30) Google Scholar)) and exists in at least two distinct quaternary conformations called R and T (12Ke H. Zhang Y. Liang J.-Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2989-2993Crossref PubMed Scopus (56) Google Scholar, 13Liu F. Fromm H.J. J. Biol. Chem. 1988; 19: 9122-9128Abstract Full Text PDF Google Scholar, 14Zhang Y. Liang J.-Y. Huang S. Lipscomb W.M. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar). AMP induces the transition from the active R-state to the inactive (or less active) T-state. Substrates or products in combination with metal cations stabilize the R- state conformation. A proposed mechanism for allosteric regulation of catalysis involves three conformational states of loop 52-72 called engaged, disengaged, and disordered (15Nelson S.W. Kurbanov F. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2001; 276: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). AMP alone or with F26P2 stabilizes a disengaged loop (16Xue Y. Huang S. Liang J.-Y. Zhang Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12482-12486Crossref PubMed Scopus (73) Google Scholar, 17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar), whereas metals with products stabilize an engaged loop (10Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar, 17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar, 18Choe J.-Y. Nelson S.W. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2003; 278: 16008-16014Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 19Choe 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). In active forms of the enzyme, loop 52-72 probably cycles between its engaged and disordered conformations (15Nelson S.W. Kurbanov F. Honzatko R.B. Fromm H.J. J. Biol. Chem. 2001; 276: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 18Choe J.-Y. Nelson S.W. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2003; 278: 16008-16014Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Fluorescence from a tryptophan reporter group at position 57 is consistent with the conformational states for loop 52-72, observed in crystal structures (20Nelson S.W. Choe J.-Y. Iancu C.V. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar, 21Wen J. Nelson S.W. Honzatko R.B. Fromm H.J. Petrich J.W. Photochem. Photobiol. 2001; 74: 679-685Crossref PubMed Scopus (5) Google Scholar). Presumably, the engaged, disengaged, and disordered conformations of loop 52-72 are possible in both the R- and T-states of FBPase, but only the engaged and disordered conformers of the R-state, and the disengaged conformer of the T-state, have been reported in crystalline complexes (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar, 18Choe J.-Y. Nelson S.W. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2003; 278: 16008-16014Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 22Ke H.M. Thorpe C.M. Seaton B. Lipscomb W.N. Marcus F. J. Mol. Biol. 1990; 212: 513-539Crossref PubMed Scopus (64) Google Scholar, 23Lu G. Stec B. Giroux E.L. Kantrowitz E.R. Protein Sci. 1996; 5: 2333-2342Crossref PubMed Scopus (23) Google Scholar). A recent report (24Wright S.W. Carlo A.A. Carty M.D. Danley D.E. Hageman D.L. Karam G.A. Levy C.B. Mansour M.N. Mathiowetz A.M. McClure L.D. Nestor N.B. McPherson R.K. Pandit J. Pustilnik L.R. Schulte G.K. Soeller W.C. Treadway J.L. Wang I.-K. Bauer P.H. J. Med. Chem. 2002; 45: 3865-3877Crossref PubMed Scopus (86) Google Scholar) in the literature identifies a new inhibitor site on FBPase, distinct from the active and the AMP-binding sites. The new family of anilinoquinazoline inhibitors was discovered by a search for potential drugs in the treatment of non-insulin-dependent diabetes. Although the site of binding on FBPase is clear, the kinetic mechanism of inhibition for this new class of inhibitor was not reported. Independent of the efforts above, a screen for new inhibitors of FBPase resulted in the discovery of a chemically distinct molecule (OC252-324, hereafter OC252, Fig. 1) that targets the same binding site on FBPase. Inhibition of FBPase by OC252 is cooperative (Hill coefficient of 2), synergistic with AMP and F26P2, noncompetitive with respect to Mg2+, but uncompetitive with respect to F16P2. OC252 greatly decreases the concentration at which substrate inhibition dominates the kinetics of FBPase. The crystal structure reveals a pair of OC252 molecules bound to each face of an FBPase tetramer. The quaternary conformation of the tetramer differs from the canonical R- and T-states, observed in the absence and presence of AMP, respectively, yet the loop (residues 52-72) is in its disengaged conformation. A strong hydrogen bond between OC252 and the side chain of a conserved aspartate residue of undetermined functional significance suggests the possibility of a binding site recognized by an unknown natural effector. Materials—F16P2, F26P2, NADP+, and AMP were purchased from Sigma. Glucose-6-phosphate dehydrogenase and phosphoglucose isomerase came from Roche Applied Science. Other chemicals were of reagent grade or the equivalent. QSW-HR high pressure liquid chromatography resin came from Toso-Hass Bioseparations. FBPase-deficient Escherichia coli strain DF 657 came from the Genetic Stock Center at Yale University. Plasmids used in the expression of wild-type FBPase came from a previous investigation (20Nelson S.W. Choe J.-Y. Iancu C.V. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar). The inhibitor OC252 was provided by Ontogen Corp. (Carlsbad, CA). Expression and Purification of Wild-type FBPase—Separate preparations of enzyme were used for the structural and kinetics investigations. Recombinant FBPase was expressed in a strain of E. coli deficient in endogenous FBPase and then purified to homogeneity. Cell-free extracts of the wild-type FBPases were subjected to heat treatment (65 °C for 5 min), followed by centrifugation. For enzyme used in kinetics investigations, the supernatant solution was loaded onto a Cibacron Blue-Sepharose column, previously equilibrated with 20 mm Tris-HCl, pH 7.5. FBPases were eluted from that column with 1 mm AMP and 20 mm Tris-HCl, pH 7.5. The eluent from the first column was loaded directly onto a DEAE-Sepharose column and then eluted with a NaCl gradient (0-0.3 m). The purified enzyme was dialyzed extensively against 20 mm Tris-HCl, pH 7.5. For crystallization experiments, after volume reduction by pressure concentration through an Amicon PM-30 membrane, the supernatant solution from centrifugation was passed through a CM-Sepharose column using a NaCl gradient (20-400 mm) in 10 mm Tris malonate, pH 6.0, and then dialyzed against KPi (20 mm, pH 7.0). Purity and protein concentrations of FBPase preparations were confirmed by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar) and the Bradford assay (26Bradford M.M. Anal. Biochem. 1976; 72: 248-252Crossref PubMed Scopus (214455) Google Scholar), respectively. Kinetic Experiments—Assays for the determination of specific activity, kcat, and activity ratios at pH 7.5 and 9.5 employed the coupling enzymes, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (1Benkovic S.T. de Maine M.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 45-82PubMed Google Scholar). The reduction of NADP+ to NADPH was monitored by absorbance spectroscopy at 340 nm. All other assays used the same coupling enzymes, but monitored NADPH production by its fluorescence emission at 470 nm, using an excitation wavelength of 340 nm. Kinetic assays were performed at room temperature (22 °C). Data fitting and analysis used the program DYNAFIT (27Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1349) Google Scholar). Crystallization of the Product Complex—Crystals of FBPase grew by the method of hanging drops. Equal parts of a protein solution (FBPase (10 mg/ml), KPi, pH 7.4 (10 mm), MgCl2 (5 mm), F6P (5 mm), and OC252 (2 mm)) and a precipitant solution (Tris malonate, pH 7.4 (2.5 mm), polyethylene glycol 3350 (6% w/v)) were combined in a droplet of 4 μl total volume. Wells contained 500 μl of the precipitant solution. Crystals of dimensions 0.4 × 0.4 × 0.3 mm grew in approximately 3 days at 20 °C. OC252 is relatively insoluble in aqueous solutions. Approximately 10 mg of pure inhibitor was dissolved initially in acetone, and appropriately measured aliquots were distributed to empty vials. The acetone was removed by evaporation, and the protein solution above was added. The inhibitor dissolved after a brief time interval to provide the clear protein solution used in the crystallization experiments. Data Collection—Data were collected at Iowa State University on a rotating anode/Siemens area detector at 120 K, using CuKα radiation passed through a graphite monochromator. Data were reduced by XENGEN (28Howard A.J. Nielsen C. Xuong N.H. Methods Enzymol. 1985; 114: 452-472Crossref PubMed Scopus (280) Google Scholar). Structure Determination, Model Building, and Refinement—Crystals grown for the present study are isomorphous to the AMP-Zn2+-product complex (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). Phase angles, used in the generation of initial electron density maps, were based on model 1EYJ of the Protein Data Bank, from which water molecules, metal cations, small molecule ligands, and residues 52-72 had been omitted. Residues 52-72 were built into the electron density of omit maps, with reference to the Cα coordinates of loop 52-72 from the AMP complex (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar), using the program XTALVIEW (29McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar) and a Silicon Graphics work station. A molecule of Pi and of F6P and two magnesium cations were added to omit electron density of the active site. Strong electron density remained, however, near the center of the tetramer, which easily accommodated a molecule of OC252. The resulting model underwent refinement, using CNS (30Brünger 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 M.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar) with force constants and parameters of stereochemistry from Engh and Huber (31Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2539) Google Scholar). A cycle of refinement consisted of slow cooling from 2500 to 300 K in steps of 25 K, followed by 120 cycles of conjugate gradient minimization, and concluded by the refinement of individual thermal parameters. Thermal parameter refinement employed restraints of 1.5 Å2 on nearest neighbor and next-to-nearest neighbor main chain atoms, 2.0 Å2 on nearest neighbor side chain atoms, and 2.5 Å2 on next-to-nearest neighbor side chain atoms. In subsequent cycles of refinement, water molecules were fit to difference electron density of 2.5σ or better and were added until no significant decrease was evident in the Rfree value. Included in the final model were water molecules that make suitable donor-acceptor distances to each other and the protein and have thermal parameters under 60 Å2. Expression and Purification of Wild-type FBPase—Expression and isolation procedures described above provide FBPase in at least 95% purity, as judged by SDS-PAGE (data not shown). The kcat value (22 ± 1 s-1) and the ratio of specific activities at pH 7.5 to 9.5 (3.3) indicate high purity and little or no proteolysis of the purified enzyme, consistent with the results from electrophoresis. Kinetics Experiments—By using fixed concentrations of MgCl2 (0.5 mm, approximately the Ka for Mg2+) and F16P2 (20 μm, saturating), initial velocities vary as the inverse square of OC252 concentration (Hill coefficient of 1.97 ± 0.1) with an I0.5 (concentration of OC252 that causes 50% inhibition) of 1.87 ± 0.07 μm (Fig. 2). F26P2, AMP, and OC252 inhibit FBPase synergistically; I0.5 values for OC252 are 0.63 ± 0.04 μm (Hill coefficient, 1.6 ± 0.1) in the presence of 1.5 μm AMP and 0.48 ± 0.01 μm (Hill coefficient, 1.7 ± 0.1) in the presence of 0.45 μm F26P2. The numerical values above result from a fit of data to Equation 1,Vo/Vm=I0.5/(I0.5-In)(Eq. 1) where n is the Hill coefficient, Vm the initial velocity in the absence of inhibitor, Vo the initial velocity at a specific inhibitor concentration, I the concentration of OC252, and I0.5 the concentration of OC252 that causes 50% inhibition. Kinetics data were taken over broad concentration ranges of Mg2+ (0.2-5 mm), F16P2 (1-20 μm), and OC252 (0-20 μm) in order to determine the kinetic mechanism of inhibition. Plots of reciprocal velocity against 1/[Mg2+]2 (F16P2 saturating, but below concentrations that cause significant substrate inhibition) and 1/[F16P2] (Mg2+ saturating) indicate noncompetitive inhibition with respect to Mg2+ and uncompetitive inhibition with respect to F16P2 (Fig. 3). As is evident from Fig. 3A, substrate inhibition increases significantly with rising inhibitor concentration. Data from Fig. 3A were fit to nine different kinetic models using the program DYNAFIT (27Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1349) Google Scholar), combining mechanisms of competitive, noncompetitive, and uncompetitive inhibition by OC252, with and without pathways for inhibitor-induced substrate inhibition or partial inhibitor-induced substrate inhibition (Table I). (Inhibitor-induced substrate inhibition requires the binding of OC252 prior to the binding of the inhibitory F16P2 molecule. Inhibitor-induced partial substrate inhibition allows turnover of the substrate-inhibited enzyme at a reduced maximal velocity.) All successful kinetic models include the association of two molecules of OC252 with an E·(Mg2+)2·F16P2 complex to form an E·(Mg2+)2·F16P2·(OC252)2 complex (uncompetitive inhibition with respect to F16P2). The latter complex induces the formation of E·(Mg2+)2·(F16P2)2·(OC252)2 (inhibitor-induced substrate inhibition). Parameters representing the formation of an E·(Mg2+)2·(OC252)2 complex (which would result in competitive, noncompetitive, or mixed inhibition with respect to F16P2 and/or the turnover of the E·(Mg2+)2·(F16P2)2·(OC252)2 complex (inhibitor-induced partial substrate inhibition) are not justified on the basis of F tests. Hence, model H of Table I represents the data at saturating Mg2+ with the fewest parameters. Scheme I then represents the kinetic mechanism of inhibition at all concentrations of the relevant ligands.Table IHypothetical models of inhibition of FBPase by OC252 Open table in a new tab Scheme IView Large Image Figure ViewerDownload Hi-res image Download (PPT) From Scheme I we derived the following relationship (Equation 2) assuming rapid-equilibrium kinetics under initial velocity conditions,1/V=(1/Vm)(1+Ka/A2+Kb/B+(KiaKb)/(A2B)+(KaI2)/(KiA2)+(I2/Kii)(1+B/Kiis))(Eq. 2) where A, B, and I are concentrations of free Mg2+, F16P2, and OC252, respectively; Vm is the maximal velocity, and Kia, Ka, Kb, Ki, Kii, and Kiis are dissociations constants for two Mg2+ atoms from the E·(Mg2+)2 complex, for two Mg2+ atoms from the E·(Mg2+)2·F16P2 complex, for F16P2 from the E·(Mg2+)2·F16P2 complex, for two OC252 molecules from the E·F16P2·(OC252)2 complex, for two molecules of OC252 from the E·(Mg2+)2·F16P2·(OC252)2 complex, and for the second (inhibitory) molecule of F16P2 from the E·(Mg2+)2·(F16P2)2·(OC252)2 complex, respectively. Equation 2 simplifies under conditions of saturating Mg2+ to Equation 3,1/V=(1/Vm)(1+Kb/B+(I2/Kii)(1+B/Kiis))(Eq. 3) which is used in the determination of Vm, Kb, Kii, and Kiis from data in Fig. 3A. Under these conditions, [F16P2]≫Kb, Equation 2 simplifies to Equation 4,1/V=(1/Vm)(1+Ka/A2+(KaI2)/(KiA2)+(I2/Kii)(1+B/Kiis))(Eq. 4) which is used in the determination of Vm, Ka, and Ki from data in Fig. 3B using numerical values for Kii and Kiis from Equation 3. Values for kinetic parameters are in Table II.Table IIKinetic parameters determined by fits of Equations 3 and 4 to the data ofFig. 3. Maximum velocity is expressed in terms of the change in fluorescence (units not defined) with timeParameterNumerical valueEquation 3Vm18.4 ± 0.3 ΔF/sKb2.16 ± 0.08 μmKii25 ± 2 μm2Kiis7.6 ± 1 μmEquation 4Vm17.1 ± 0.8 ΔF/sKa0.38 ± 0.02 mm2Ki3.65 ± 0.6 μm2 Open table in a new tab OC252 Complex of FBPase (Protein Data Bank Code 1Q9D)—Crystals grown in the presence of OC252 belong to the space group P21212 (a = 58.86, b = 166.25, c = 80.27), and are isomorphous to those of AMP complexes of FBPase (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). Two subunits of FBPase are in the asymmetric unit of this crystal form. Cα coordinates of the independent subunits superimposed with a root mean square deviation of 0.38 Å, using a subset of residues (33-49, 75-265, and 272-330), which do not exhibit significant tertiary conformational differences between the T- and R-state subunits of FBPase (10Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar). Regions of weak or absent electron density include residues 1-8 and 63-70. The model begins at residue 9 and continues to the last residue of the sequence, but segment 63-70 is unreliable, as evidenced by high thermal parameters. Thermal parameters vary from 5 to 57 Å2. As determined by PROCHECK (32Laskowski R.A. Mac Arthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), the model has stereochemistry generally comparable with that of structures derived from data of nominal resolution 2.0 Å. Statistics for data collection and refinement are in Table III, and an overview of the complex appears in Fig. 4.Table IIIStatistics of data collection and refinement for the OC252-FBPase complexResolution (Å)2.35No. measurements94,949No. unique reflections28,849Completeness of dataOverall87.5Last shell (2.44-2.35 Å)65.5RsymaRsym=ΣjΣi|Iij-〈Ij〉|/ΣiΣjIij, where i runs over multiple observations of the same intensity and j runs over crystallographic unique intensities.5.9No. reflections in refinementbAll data are in the resolution range of 50-2.35 Å.27,163No. atoms5434No. solvent sites290R-factorcR-factor=Σ||Fobs|-|Fcalc||/Σ|Fobs|, where|Fobs| > 0.0.191RfreedR-factor based upon 10% of the data randomly culled and not used in the refinement.0.246Mean thermal parameters(Å2)Protein21.1Mg2+22.1F6P24.5Pi50.1OC25213.6Root mean square deviationsBond lengths (Å)0.012Bond angles (degree)1.7Dihedral angles (degree)24.1Improper angles (degree)5.74a Rsym=ΣjΣi|Iij-〈Ij〉|/ΣiΣjIij, where i runs over multiple observations of the same intensity and j runs over crystallographic unique intensities.b All data are in the resolution range of 50-2.35 Å.c R-factor=Σ||Fobs|-|Fcalc||/Σ|Fobs|, where|Fobs| > 0.d R-factor based upon 10% of the data randomly culled and not used in the refinement. Open table in a new tab Although the two subunits of the OC252 complex are similar, the ligation of each active site by metals differs. In the subunit labeled chain B, strong electron density is at metal site 1. The thermal parameter of Mg2+ at full occupancy here refines to a value of 10 Å2, which is significantly less than the average value (20 Å2) for atoms of the protein ligated to that metal. Hence Mg2+ and a heavier metal (Zn2+, on the basis of prior experience (19Choe 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)) probably co-occupy site 1 in chain B. Site 2 of chain B has diffuse electron density that may represent disordered water molecules or the combination of water molecules and Mg2+ at low occupancy; Mg2+ at full occupancy refined at metal site 2 with a thermal parameter of 32 Å2. In the subunit labeled chain A, Mg2+ refines with thermal parameters of 18 and 29 Å2 at sites 1 and 2, respectively. Metal site 2 in chain A may be occupied partially by Mg2+. The conformations of side chains near metal site 2 also differ in each of the symmetry unique subunits of the crystal. In addition, molecules of Pi are at partial occupancy at each active site, but molecules of F6P are at full occupancy. OC252 is arguably a highly constrained pseudo-tetrapeptide (Fig. 1); the first and fourth “residues” have tyrosyl side chains, a methylene group in residue 2 covalently bridges the phenylalanyl side chain to its amide nitrogen, and an n-propyl group is the side chain of residue 3. The N-2 atom of OC252 corresponds to the backbone nitrogen atom of the first and third residues, and the terminal carboxyl group is absent. Atoms C-8, C-10, and C-12 have the l-configuration of a common amino acid. In their FBPase complex, two molecules of OC252 are in mutual contact, related to each other by the molecular 2-fold axis that projects through the face of the FBPase tetramer. Most of the contacts between inhibitor and protein are apolar; however, the phenol oxygen atoms of residues 1 and 4 of OC252 hydrogen-bond with backbone carbonyl 71 and the side chain of Asp187, respectively. Furthermore, the C—O bond axis of backbone carbonyl 51 is normal to the plane defined by atoms C-8, N-2, C-11, and C-12 of OC252, with its oxygen atom 3.2 Å from atom N-2. The latter suggests an electronic resonance state of the bound OC252 molecule that stabilizes this close contact (Fig. 1). Although the OC252 and AMP crystalline complexes are isomorphous, the two FBPase tetramers adopt different quaternary conformations. Superpositions of C1-C2 subunit pairs from the OC252, T-state, and R-state complexes give comparable root mean squared deviations (Table IV). (See Fig. 4 for the convention adopted in the labeling of subunits.) On the other hand, superpositions of tetramers give significantly larger deviations, suggesting different quaternary structures. Indeed, the C3-C4 subunit pair in the OC252 complex rotates ∼14° relative to the C1-C2 subunit pair, falling some 3° short of the canonical T-state (Fig. 5). Hereafter, we refer to the quaternary state of FBPase induced by OC252 as the I-state.Table IVRoot mean squared deviations (in Å) in superpositions of C1-C2 subunits and tetramers (boldface) of FBPaseT-stateOC252 complexR-state2.71/0.5782.16/0.481T-state0.703/0.332 Open table in a new tab The I-state must be due to the specific effects of the OC252 inhibitor. First, the conditions of crystallization for the T-, R-, and I-states of FBPase differ only by the presence or absence of allosteric effectors. The T-state crystallizes in the presence of AMP (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar), the I-state in the presence of OC252, and the R-state in the absence of allosteric effectors (10Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar, 17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). Hence, ligation of FBPas
