Inhibition of Fructose-1,6-bisphosphatase by a New Class of Allosteric Effectors

Abstract

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 of the wild-type were to treatment for by enzyme used in kinetics the was a with were from that with AMP and The from the was a and then with a The purified enzyme was by pressure concentration an the from was a a in and then and concentrations of FBPase preparations were by PubMed Scopus Google Scholar) and the M.M. Biochem. PubMed Scopus Google Scholar), for the of and at and employed the phosphoglucose isomerase and dehydrogenase (1Benkovic S.T. de Maine M.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 45-82PubMed Google Scholar). The of to was by at used the same but by its at an of were at and used the Biochem. 1996; PubMed Scopus Google Scholar). of the of FBPase by the of of a and OC252 and a were in a of of the of in at OC252 is relatively in of inhibitor was in and were to The was by and the was The inhibitor a to the used in the were at on a at a were by A.J. Enzymol. PubMed Scopus Google Scholar). and for the are to the complex (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). used in the of were on of the Protein from which metal molecule and 52-72 been 52-72 were the of with to the 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), the D.E. J. Mol. 10: Google Scholar) and a A molecule of Pi and of and two cations were to of the active near the center of the tetramer, which a molecule of The J. Biol. 1998; 54: PubMed Scopus Google Scholar) with and of from and A. 1991; Scopus Google Scholar). A of of from to in of by cycles of and by the of employed of on nearest and chain on nearest side chain and on side chain In cycles of molecules were to of or and were was in the in the were molecules that to each and the and have Expression and Purification of Wild-type and FBPase in at least as by not The and the of at to high and or of the purified enzyme, consistent with the from concentrations of the for and F16P2 as the of OC252 concentration (Hill coefficient of with an of OC252 that of F26P2, AMP, and OC252 inhibit FBPase for OC252 are (Hill in the presence of AMP and (Hill in the presence of The from a of to 1) is the the in the absence of the at a inhibitor the concentration of OC252, and the concentration of OC252 that were concentration of F16P2 and OC252 in order to the kinetic mechanism of of but concentrations that substrate and noncompetitive inhibition with respect to and uncompetitive inhibition with respect to F16P2 is from Fig. substrate inhibition with inhibitor from Fig. were to kinetic the Biochem. 1996; PubMed Scopus Google Scholar), of and uncompetitive inhibition by OC252, with and for substrate inhibition or substrate inhibition substrate inhibition the binding of OC252 to the binding of the inhibitory F16P2 substrate inhibition of the enzyme at a kinetic the of two molecules of OC252 with an complex to an complex inhibition with respect to The complex induces the of substrate the of an complex in or inhibition with respect to F16P2 the of the complex substrate are not on the of Hence, of the at with the then the kinetic mechanism of inhibition at all concentrations of the of inhibition of FBPase by OC252 in a new the kinetics and are concentrations of Mg2+, and OC252, is the and and are for two from the for two from the for F16P2 from the for two OC252 molecules from the for two molecules of OC252 from the and for the molecule of F16P2 from the of to which is used in the of and from in Fig. to which is used in the of and from in Fig. for and from for kinetic are in by of and to the is expressed in of the in not with in a new OC252 of FBPase in the presence of OC252 to the group and are to of AMP complexes of FBPase (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). subunits of FBPase are in the of this crystal of the subunits with a of a of and which not conformational between the 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). of or and The at residue and to the residue of the but is as by high from to 57 by J. Google Scholar), the with that of structures from of for and are in and an of the complex in Fig. of and for the of of the same and in are in the of of the and not used in the of the same and are in the of of the and not used in the in a new Although the two subunits of the OC252 complex are the of each active site by metals In the chain strong is at metal site The of at to a of which is less the for of the ligated to that and a metal on the of 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 site in chain B. of chain that may disordered molecules or the combination of molecules and at at at metal site with a of In the chain with of and at and site in chain A may by The conformations of side near metal site in each of the subunits of the In molecules of Pi are at at each active but molecules of are at OC252 is a highly constrained pseudo-tetrapeptide the and have side a group in residue the side chain to its and an group is the side chain of residue The of OC252 to the backbone of the and and the group is and have the of a In FBPase two molecules of OC252 are in to each by the that the face of the FBPase tetramer. of the between inhibitor and are the of and of OC252 with backbone carbonyl and the side chain of the bond of backbone carbonyl is to the by and of OC252, with its Å from The suggests an state of the bound OC252 molecule that stabilizes this contact Although the OC252 and AMP crystalline complexes are the two FBPase quaternary of pairs from the OC252, T-state, and R-state complexes Fig. for the in the of the of quaternary the pair in the OC252 complex to the of the canonical T-state to the quaternary state of FBPase by OC252 as the in of subunits and of in a new The to the of the OC252 inhibitor. the of for the and of FBPase only by the presence or absence of allosteric The T-state 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 in the presence of OC252, and the R-state in the absence of allosteric (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, of FBPase by OC252 the OC252, stabilizes the disengaged conformation of loop 52-72, which to this been only in Moreover, of the side chain of its bond the only contact between OC252 and the T-state. Hence, OC252 could bind to T-state FBPase in an the of the engaged loop conformation observed only in the and the side chain of the the OC252 binding in the OC252 may bind to the R-state enzyme, but at the least loop 52-72 in its engaged conformation. Although loop 52-72 the conformational at the between subunits in the and of FBPase, in the of the AMP and are Å to each in the T-state in the R-state, a to the binding of AMP Y. Liang J.-Y. Huang S. Lipscomb W.M. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar). of and T-state a more complex to the T-state, the binding site for the of AMP is in the The of backbone and with the group of AMP the an contact with the of a conformational in the two of this contact but in and The side of and are in contact with of a in with the group of the of and the AMP then is in the the T-state then hydrogen of the group of AMP to conformational at the OC252 and (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) a site on FBPase, but the binding of inhibitors Each binds as a but bound anilinoquinazoline and OC252 molecules have two molecules distinct of a OC252 molecule The of the anilinoquinazoline and of OC252, whereas the residue of the OC252 of the anilinoquinazoline molecule to residue of OC252, the side chain of which In the the side chain of the by the side chain of OC252 residue The anilinoquinazoline is the of the contact the of the FBPase tetramer with the of the side and side of OC252, on the the of the tetramer. Hence, the of between OC252 and FBPase greatly that of the Each OC252 molecule interacts with from three of four subunits of FBPase, whereas each anilinoquinazoline molecule interacts with from a pair of subunits all the above, both inhibitor with backbone carbonyl of FBPase. the the group of the with backbone carbonyl whereas of the OC252 molecule is in a possible contact with the of the backbone carbonyl 51. of wild-type are competitive as or noncompetitive as with respect to F16P2. inhibition of FBPase by OC252 with respect to the substrate is but not The of to the kinetic mechanism of AMP inhibition with respect to F16P2 from noncompetitive to but the competitive mechanism of AMP inhibition with respect to Poland B.W. Honzatko R.B. Fromm H.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). AMP inhibits FBPase at concentrations of and at high concentrations of F16P2. The side chain of in in the T-state of FBPase, and but is less in the R-state, of (17Choe J.-Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). The of by then may the T-state to the AMP may bind to FBPase in the R-state but not a transition to the T-state. F16P2 could stabilize an state of FBPase that the binding of AMP, and F16P2 and AMP in the absence of metals bind to wild-type FBPase M.M. El-Maghrabi M.R. Pilkis S.J. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar). uncompetitive of inhibition then AMP of this state a transition to the T-state. The quaternary state of FBPase by OC252 may to this state of FBPase. uncompetitive inhibition by OC252 with respect to F16P2 binding of the active site by F16P2 enhances the of OC252 for its binding site Moreover, the binding of OC252 induces F16P2 inhibition between OC252 and F16P2 each ligand were to bind at distinct and stabilize a conformational state of FBPase. Hence, the observed in the OC252 complex may to the quaternary state of FBPase in the presence of inhibitory concentrations of of FBPase by OC252 the engaged conformation of loop 52-72, which is for The may the engaged conformation of loop 52-72, for the of substrate inhibition of FBPase at concentrations of F16P2 in of The may the for synergistic inhibition of FBPase by OC252 and Elevated of F26P2 a transition from the R-state to a conformation but not identical to the T-state, as by from a reporter group (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). high concentrations of F26P2 stabilize the and the tetramer the in a crystal structure of an Choe and B. OC252 and F26P2 stabilize the same quaternary conformation by at distinct sites, functional in of the between AMP and OC252 is only more OC252 the engaged conformer of loop 52-72 and stabilizes the disengaged loop in the AMP complex of FBPase. Moreover, the probably on or near the the R- and by the OC252 of the that AMP in the R- to T-state is the of FBPase an for the of new of the concentrations of F16P2 in in Scholar)) are that substrate inhibition and of FBPase. concentrations of F26P2 and products that stabilize the in are in of in the AMP and F26P2 are the of FBPase. that OC252 inhibits FBPase with AMP and F26P2 at concentrations of the allosteric of FBPase could an in not the used to screen for potent inhibition of FBPase have employed concentrations of F16P2 as high as (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). inhibitors that with F16P2 and the screen in of that inhibit FBPase only in the presence of bound F16P2. Although this to the discovery of molecules that not bind to the active it the of that high concentrations of F16P2 for potent is for OC252 in the presence of F16P2 but to at a concentration of F16P2. Hence, an screen high concentrations of F16P2 may new that the allosteric site of FBPase, but further kinetic at concentrations of F16P2 may more in the as a potent inhibitor in The that inhibition from the of the allosteric of FBPase. an may of FBPase Asp187 as an The of Asp187 to only a on the functional of FBPase G. Giroux E.L. Kantrowitz E.R. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar). to on FBPase that to the Iancu and B. pressure then aspartate at position and is it that OC252 to this side The of Asp187 suggests a in the of a natural effector. S. E. S. Proc. Natl. Acad. Sci. U. S. A. 1980; PubMed Scopus Google Scholar, S. E. Biochem. 1983; PubMed Scopus (5) Google Scholar, A. Biochem. 2000; PubMed Scopus (30) Google Scholar, W.C. Biochem. 1994; PubMed Scopus Google Scholar) are in the for between FBPase and The of are essential to and the for both A and of in which residue of Hence, a potential may have to with a natural effector for the allosteric or FBPase may as an effector in of may to the of efforts to drugs that FBPase.