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• W2040300526 abstract "Residues 1–10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reduceskcat by 30-fold and Mg2+ affinity by 20-fold and eliminates cooperativity in Mg2+ activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mm. Deletion of the first seven residues reduces kcat and Mg2+ affinity significantly but has no effect on AMP inhibition. The mutation of Asp9 to alanine reproduces the weakened affinity for Mg2+ observed in the deletion mutants, and the mutation of Ile10 to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52–72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast. Residues 1–10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reduceskcat by 30-fold and Mg2+ affinity by 20-fold and eliminates cooperativity in Mg2+ activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mm. Deletion of the first seven residues reduces kcat and Mg2+ affinity significantly but has no effect on AMP inhibition. The mutation of Asp9 to alanine reproduces the weakened affinity for Mg2+ observed in the deletion mutants, and the mutation of Ile10 to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52–72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast. fructose-1,6-bisphosphatase fructose 1,6-bisphosphate fructose 2,6-bisphospate orthophosphate circular dichroism 2′-(or 3′)-O-(trinitrophenyl) adenosine 5′-monophosphate Fructose-1,6-bisphosphatase (d-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase1) catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose 6-phosphate and inorganic phosphate (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, 3Van Schaftingen E. Adv. Enzymol. Relat. Areas Mol Biol. 1987; 59: 45-82Google Scholar). The reaction facilitated by FBPase is subject to hormone and metabolite regulation, the net result of which is the tight coordination of FBPase and fructose 6-phosphate 1-kinase activities (4Pilkis S.J. el-Maghrabi M.R. Claus T.H. Annu. Rev. Biochem. 1988; 57: 755-783Crossref PubMed Scopus (317) Google Scholar). FBPase activity requires divalent cations such as Mg2+, Mn2+, or Zn2+, and plots of velocity versus metal ion concentration are sigmoidal with a Hill coefficient of 2.0 (5Nimmo H.G. Tipton K.F. Eur. J. Biochem. 1975; 58: 567-574Crossref PubMed Scopus (50) Google Scholar, 6Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar, 7Scheffler J.E. Fromm H.J. Biochemistry. 1986; 25: 6659-6665Crossref PubMed Scopus (32) Google Scholar). AMP binds cooperatively (Hill coefficient of 2) 28 Å from the nearest active site (8Xue Y. Huang S. Liang J.Y. Zhang Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 99: 12482-12486Crossref Scopus (73) Google Scholar) and inhibits the enzyme, whereas F26P2binds at the active site (9Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar). Inhibition of FBPase by AMP and F26P2 is synergistic. F26P2 can lower the apparent inhibition constant for AMP by up to 10-fold (6Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar).FBPase is a tetramer of identical subunits (Mr = 37,000). To a first approximation, these subunits lie in the same plane and occupy the corners of a square in one of the principal quaternary states of the mammalian enzyme (R-state). By past convention, subunit C1 occupies the upper left-hand corner, with subunits C2–C4 following in a clockwise direction. AMP causes a transition from the R-state to the T-state, driving a 17° rotation of the C1-C2 subunit pair with respect to the C3-C4 pair about a molecular 2-fold axis of symmetry (10Shyur 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). Complexes of FBPase with AMP in the presence of F16P2, F26P2, and fructose 6-phosphate are all in the T-state (9Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar, 11Ke H.M. Zhang Y.P. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5243-5247Crossref PubMed Scopus (119) Google Scholar, 12Villeret V. Huang S. Zhang Y. Lipscomb W.N. Biochemistry. 1995; 34: 4307-4315Crossref PubMed Scopus (51) Google Scholar), whereas in the absence of AMP, the enzyme has appeared in the R-state in crystal structures (13Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar, 14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar).Mutations in loop 52–72 and in the hinge preceding the loop (residues 50–51) greatly influence catalysis and AMP inhibition of FBPase and together suggest the necessity of an engaged conformation for loop 52–72 for catalysis under physiological conditions (15Kurbanov 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, 16Nelson 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). The engaged conformation, in which loop 52–72 interacts with the active site, occurs in metal-product complexes of wild-type FBPase but only in the absence of AMP (17Nelson S.W. Choe J.-Y. Iancu C.V. Honzatko R.B. Fromm H.J. Biochemistry. 2000; 39: 11100-11106Crossref PubMed Scopus (26) Google Scholar). All residues of the engaged loop are in well defined conformations associated with clear electron density in crystal structures (13Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar, 14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). The disengaged conformation of loop 52–72 exists in AMP complexes of wild-type FBPase. The disengaged loop is far from the active site. Residues 52–60 lie near, and interact with, an adjacent subunit (C1-C2 interaction), but residues 61–73 are disordered, with no observable electron density in crystal structures (14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). In FBPase crystallized without metal cations (18Ke H. Thorpe C.M. Seaton B.A. Marcus F. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1475-1479Crossref PubMed Scopus (55) Google Scholar) and in certain mutant forms of FBPase (to be discussed below in greater detail), loop 52–72 exists in yet another conformation in which residues 54–73 are without electron density. This disordered state of the loop 52–72 is a manifold of closely related conformations in which the loop itself interacts weakly with the rest of the enzyme.AMP binds between helices H1 and H2 of FBPase and causes modest movements in both helices relative to the rest of the subunit (14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). The 0.9-Å movement of helix H2 along its axis directly influences loop 52–72 (13Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar), whereas the 1.5-Å movement of helix H1 could influence loop 52–72 of an adjacent subunit (14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). AMP may stabilize the disengaged conformation of loop 52–72 through helix H1 by facilitating contacts between residues 52–59 of the loop and residues 1–10 of the N terminus. Residues belonging to this N-terminal segment are either disordered (residues 1–6) or exist in different conformations in the R- and T-state of FBPase (residues 7–10). In addition, several of these N-terminal residues are conserved throughout FBPases from eukaryotes, suggesting a functional significance, as yet unconfirmed.Here we present the changes in functional properties of FBPase due to deletion and point mutations in the N-terminal segment (residues 1–10). Mutations, which directly or indirectly influence positions 9 and 10, cause large perturbations in catalysis and/or AMP inhibition. A simple model in which the quaternary states of FBPase differentially stabilize specific conformations of loop 52–72 accounts for the properties of wild-type and mutant FBPases presented here and in other studies.DISCUSSIONCrystal structures of FBPase from mammalian sources and the chloroplast have little or no electron density for the N-terminal region of the enzyme. Yet certain amino acids in this region, particularly residues 9–10, are conserved over a wide range of organisms. As porcine FBPase has no tryptophan, incorporation of a tryptophan for Phe6 introduces a unique spectroscopic probe. The functional properties of Phe6 → Trp FBPase are comparable with those of the wild-type enzyme, and fluorescence emission in the presence and absence of AMP is about the same. Evidently, the first six residues of porcine FBPase have no functional role in catalysis or metabolite regulation of catalysis.Position 7 may be the first residue from the N terminus to directly or indirectly influence the functional properties of FBPase. Asp7 is the first residue defined by electron density in crystal structures of wild-type FBPase (14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar). The 7DEL construct exhibits significant changes in functional properties (Table I), in particular Ka-Mg2+ is elevated 15-fold relative to that of wild-type FBPase. Asp7 → Ala FBPase, however, has functional properties similar to those of the wild-type enzyme. Hence, the functional changes due to the 7DEL construct may be the consequence of an indirect perturbation on the conformation of residues 9–10 and/or the conformational properties of loop 52–72 in the R-state (AMP absent). Interestingly, the mutation of the Lys50 to proline (a hinge element for loop 52–72) eliminates AMP inhibition by the disruption of the allosteric mechanism and also causes a 15-fold elevation inKa-Mg2+ (16Nelson 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). Other mutations in the segment 50–55 cause little effect on AMP inhibition but increaseKa-Mg2+ by 10-fold or more. 2C. Iancu, H. J. Fromm, and R. B. Honzatko, unpublished data. The impact of the 10DEL construct on the functional properties of FBPase is even more extreme than is that of the 7DEL mutant. The low pH activity ratio is consistent with the effects of the mutation of Lys50 to Pro (16Nelson 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) and of the truncation of 25 residues from the N terminus by proteolysis of FBPase (24Chatterjee T. Reardon I. Heinrikson R.L. Marcus F.J. J. Biol. Chem. 1985; 260: 13359-13553Google Scholar). The low pH-activity ratio could result from the proteolysis of loop 52–72 (25Horecker B.L. Melloni E. Pontremoli S. Adv. Enzymol. Relat. Areas Mol. Biol. 1975; 42: 193-226PubMed Google Scholar); however, N-terminal sequencing of the 10DEL mutant excludes this possibility. Instead, the 15-fold increase in Ka-Mg2+and the 30-fold decrease in kcat is consistent with the failure of loop 52–72 to achieve an engaged conformation in the R-state of the 10DEL construct. The effects of the 10DEL mutant can be explained in part by the side chain at position 9. Asn9→ Ala FBPase exhibits a 10-fold increase in itsKa-Mg2+ but no change inkcat or the pH-activity ratio relative to the wild-type enzyme. In R-state crystal structures, Asn9hydrogen bonds with Arg15 (Fig.4). The Asn9-Arg15 interaction probably stabilizes residues 7–11 in their R-state conformation. These residues pack between helix 3 and loop 187–194. Loop 187–194 interacts through hydrogen bonds and nonbonded contacts with hinge residues preceding loop 52–72 (Fig. 4). The loss of the Asn9-Arg15 hydrogen bond may lead to a long range conformational change, which destabilizes the engaged conformation of loop 52–72. Indeed, the disordered conformation of loop 52–72 appears hand in hand with disorder in residues 1–11 in R-state crystal structures (9Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar, 18Ke H. Thorpe C.M. Seaton B.A. Marcus F. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1475-1479Crossref PubMed Scopus (55) Google Scholar).Unlike the 7DEL construct, which has little effect on allosteric inhibition by AMP, the 10DEL mutation significantly alters the maximum level of AMP inhibition. The 25-residue truncated form of FBPase is not inhibited by AMP (24Chatterjee T. Reardon I. Heinrikson R.L. Marcus F.J. J. Biol. Chem. 1985; 260: 13359-13553Google Scholar). As some of the 25 residues are part of the AMP binding pocket (14Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (63) Google Scholar), the effect of limited proteolysis is of little surprise. In contrast, the first 10 residues are remote from the site of AMP binding, and indeed, AMP-PNP binds with nearly identical affinity constants to 10DEL and wild-type FBPases. Changes in the functional properties due to the 10DEL mutant, then, must result from perturbations in the allosteric mechanism of AMP inhibition. Although no single point mutation reproduced the functional properties of the 10DEL construct in the absence of AMP, the mutation of Ile10 to aspartate reproduced the phenomenon of 50% maximal inhibition by AMP. As noted below, the hydrophobic side chain at position 10 is critical to the stability of the disengaged conformation of loop 52–72 in the T-state.The following model is a basis for understanding AMP inhibition in wild-type FBPase and in mutant FBPases. Hereafter, T-state and R-state refer to quaternary arrangements of subunits in FBPase, distinguished by the 17° rotation about a molecular symmetry axis. In addition, the subunits in the T- and R-states can adopt different tertiary conformations, the most significant of which involve conformational changes in loop 52–72. In the T-state of wild-type FBPase, loop 52–72 is in the disengaged conformation. The T-state subunit arrangement stabilizes the disengaged conformation by interactions, which involve residues 50–60 of subunit C1 with residues 187–194 and 9–11 of subunit C2 (Fig. 5). Point mutations in these structural elements profoundly influence AMP inhibition (16Nelson 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, 26Lu G. Giroux E.L. Kantrowitz E.R. J. Biol. Chem. 1997; 272: 5076-5081Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,27Carcamo J.G. Yanez A.J. Ludwig H.C. Leon O. Pinto R.O. Reyes A.M. Slebe J.C. Eur. J. Biochem. 2000; 267: 2242-2251Crossref PubMed Scopus (15) Google Scholar), AMP cooperativity (26Lu G. Giroux E.L. Kantrowitz E.R. J. Biol. Chem. 1997; 272: 5076-5081Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 27Carcamo J.G. Yanez A.J. Ludwig H.C. Leon O. Pinto R.O. Reyes A.M. Slebe J.C. Eur. J. Biochem. 2000; 267: 2242-2251Crossref PubMed Scopus (15) Google Scholar), F26P2 inhibition (16Nelson 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, 27Carcamo J.G. Yanez A.J. Ludwig H.C. Leon O. Pinto R.O. Reyes A.M. Slebe J.C. Eur. J. Biochem. 2000; 267: 2242-2251Crossref PubMed Scopus (15) Google Scholar), and/or metal affinity (16Nelson 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). The R-state subunit arrangement does not stabilize the disengaged loop conformation. As a consequence loop 52–72 occupies an engaged conformation or a disordered conformation. The engaged conformation was first observed in the context of a product-Zn2+ complex of the wild-type enzyme (13Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. Biochemistry. 1998; 33: 11441-11450Crossref Scopus (59) Google Scholar). The disordered conformation of loop 52–72 appears in FBPase structures crystallized in the absence of metal activators (18Ke H. Thorpe C.M. Seaton B.A. Marcus F. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1475-1479Crossref PubMed Scopus (55) Google Scholar).Figure 5Structural elements important to allosteric regulation in the T-state of FBPase. Shown here are segments of two polypeptide chains (residues 9–89) viewed down an axis of molecular 2-fold symmetry toward the interface buried between subunits C1-C2 and subunits C3-C4 (top). The side chain of Ile10 from subunit C2 makes nonbonded contacts with Thr12 and Ile194, both from subunit C2, and Tyr57 and Ile59, both from loop 52–72 of subunit C1 (bottom). Orientations of top and bottom illustrations are the same. This illustration was drawn with MOLSCRIPT (33Kraulis J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Catalysis occurs at neutral pH if loop 52–72 can cycle between its engaged and disordered conformations. A loop that is always engaged is a dead-end complex. A loop that cannot achieve the engaged conformation results in low metal affinity and little or no activity at neutral pH. Assuming loop 52–72 exchanges between its engaged and disordered conformations, the free energy differences between these conformational states must be small, that is, the R-state maintains small free-energy differences between disordered and engaged conformations of loop 52–72. In contrast, the T-state subunit arrangement selectively stabilizes a new conformation for loop 52–72 (disengaged conformation), which de-populates the disordered/engaged loop conformations. The decline in the catalytic rate of T-state FBPase is directly related to the differences in free energy between the engaged/disordered loop conformations and the disengaged loop conformation. For the wild-type enzyme, the free energy difference is large; hence, little or no catalysis occurs. For specific mutants of FBPase that selectively destabilize the disengaged loop conformation, the free energy difference is less, the engaged/disordered loop conformations are more populated, and hence, a measurable level of catalysis occurs. Biphasic AMP inhibition should appear whenever a mutation selectively destabilizes the disengaged loop conformation of the T-state. FBPase mutants with biphasic AMP inhibition (Lys42 → Ala, Glu192 → Ala, Glu192 → Gln, Lys50 → Asn, Lys50 → Pro/Tyr57 → Trp, and Ile10 → Asp) exhibit plateau activities, which vary from 10 to 70% full activity (16Nelson 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, 26Lu G. Giroux E.L. Kantrowitz E.R. J. Biol. Chem. 1997; 272: 5076-5081Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 27Carcamo J.G. Yanez A.J. Ludwig H.C. Leon O. Pinto R.O. Reyes A.M. Slebe J.C. Eur. J. Biochem. 2000; 267: 2242-2251Crossref PubMed Scopus (15) Google Scholar). As suggested by Kantrowitz and co-workers (28Lu G. Stec B. Giroux E.L. Kantrowitz E.R. Protein Sci. 1996; 5: 2333-2342Crossref PubMed Scopus (23) Google Scholar), the activity most likely comes from FBPase in the T-state subunit arrangement. Here we add that some fraction of the mutant FBPases have engaged/disordered loop conformations in the presence of AMP, which are responsible for the observed turnover. The free energy relationships of the model are summarized in Fig.6.Figure 6Changes in relative free energy levels of three loop conformations in the R- and T-states of wild-type and Asp10 FBPases. Bold lines indicate the energies of the engaged and disengaged conformations of loop 52–72, whereas the bundle of fine lines represents the disordered loop, which is in a manifold of conformational states with nearly equivalent free energies. In the wild-type enzyme, the R to T transition selectively stabilizes the disengaged conformation of loop 52–72 over the engaged and disordered conformational states. In the Ile10 → Asp mutant, the T-state does not stabilize a disengaged conformation for loop 52–72. The separation in free energy between the engaged conformation and the disordered conformation in the T-state (ΔG in figure) may influence the observed level of catalytic activity in the presence of saturating AMP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)From the model above, mutant FBPases with biphasic AMP inhibition must have a less stable, disengaged conformation of loop 52–72 in the T-state. In fact, structural evidence supports this claim. The disengaged loop in the T-state of wild-type FBPase has interpretable electron density up to residue 60, with the residues 50–60 participating in interactions across the C1-C2 subunit interface(14). The mutation of Lys42 to alanine results in biphasic AMP inhibition with 70% relative activity at the plateau (26Lu G. Giroux E.L. Kantrowitz E.R. J. Biol. Chem. 1997; 272: 5076-5081Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The crystal structure of the AMP complex of the Lys42→ Ala mutant is in the T-state, but loop 52–72 is poorly ordered from residues 54 to 72 (28). In the crystal structure of the AMP complex of Ile10 → Asp2 the enzyme is again in the T-state, and loop 52–72 is without electron density from residues 54 to 72. Hence, loop 52–72 in the Ala42 and Asp10 mutants is not in the disengaged conformation of wild-type FBPase but, rather, in a disordered conformation similar to that of the R-state apoenzyme. If the disengaged structure is de-populated in favor of disordered conformations, then perhaps under the appropriate conditions an engaged conformation for loop 52–72 may occur in the T-state of such mutants.The thioredoxin-mediated formation of a disulfide bond between Cys153 and Cys173 in chloroplast FBPase (29Jacquot J.P. Lopez-Jaramillo J. Miginiac-Maslow M. Lemaire S. Cherfils J. Chueca A. Lopez-Gorge J. FEBS Lett. 1997; 401: 143-147Crossref PubMed Scopus (80) Google Scholar) putatively stabilizes the position of a loop that excludes the binding of metal activators and the engaged conformation of loop 61–81 (which corresponds to loop 52–72 of mammalian FBPase) (30Chiadmi M. Navaza A. Miginiac-Maslow M. Jacquot J.P. Cherfils J. EMBO J. 1999; 18: 6809-6815Crossref PubMed Scopus (104) Google Scholar). FBPase from the chloroplast evidently does not bind AMP (30Chiadmi M. Navaza A. Miginiac-Maslow M. Jacquot J.P. Cherfils J. EMBO J. 1999; 18: 6809-6815Crossref PubMed Scopus (104) Google Scholar, 31Nel W. Terblanche S.E. Int. J. Biochem. 1992; 24: 1267-1283Crossref PubMed Scopus (9) Google Scholar). Yet existing crystal structures of reduced and oxidized chloroplast FBPase have nearly the same quaternary arrangement of subunits, resembling most closely the T-state of mammalian FBPase. Hence, as illustrated by FBPase from the chloroplast, a T-state subunit arrangement by itself does not exclude catalysis. Interestingly, position 18 of the chloroplast enzyme, which corresponds to position 10 of mammalian FBPase, is isoleucine, this residue being widely conserved among FBPases. Furthermore, in the inactive, oxidized form of chloroplast FBPase, loop 61–81 (corresponding to loop 52–72 of the mammalian enzyme) is in the disengaged conformation (30Chiadmi M. Navaza A. Miginiac-Maslow M. Jacquot J.P. Cherfils J. EMBO J. 1999; 18: 6809-6815Crossref PubMed Scopus (104) Google Scholar), whereas the equivalent loop in the active, reduced form of chloroplast FBPase is in the disordered conformation (32Villeret V. Huang S. Zhang Y. Xue Y. Lipscomb W.N. Biochemistry. 1995; 34: 4299-4306Crossref PubMed Scopus (73) Google Scholar). Evidently, oxidation and reduction of the disulfide bond in chloroplast FBPase influences the relative stability of the disengaged loop-conformation in a manner similar to that of AMP in mammalian FBPases. The observation of corresponding conformational states in mammalian and chloroplast FBPases infers a common, early mechanism of FBPase regulation that has diverged through evolution. Fructose-1,6-bisphosphatase (d-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase1) catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose 6-phosphate and inorganic phosphate (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, 3Van Schaftingen E. Adv. Enzymol. Relat. Areas Mol Biol. 1987; 59: 45-82Google Scholar). The reaction facilitated by FBPase is subject to hormone and metabolite regulation, the net result of which is the tight coordination of FBPase and fructose 6-phosphate 1-kinase activities (4Pilkis S.J. el-Maghrabi M.R. Claus T.H. Annu. Rev. Biochem. 1988; 57: 755-783Crossref PubMed Scopus (317) Google Scholar). FBPase activity requires divalent cations such as Mg2+, Mn2+, or Zn2+, and plots of velocity versus metal ion concentration are sigmoidal with a Hill coefficient of 2.0 (5Nimmo H.G. Tipton K.F. Eur. J. Biochem. 1975; 58: 567-574Crossref PubMed Scopus (50) Google Scholar, 6Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar, 7Scheffler J.E. Fromm H.J. Biochemistry. 1986; 25: 6659-6665Crossref PubMed Scopus (32) Google Scholar). AMP binds cooperatively (Hill coefficient of 2) 28 Å from the nearest active site (8Xue Y. Huang S. Liang J.Y. Zhang Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 99: 12482-12486Crossref Scopus (73) Google Scholar) and inhibits the enzyme, whereas F26P2binds at the active site (9Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar). Inhibition of FBPase by AMP and F26P2 is synergistic. F26P2 can lower the apparent inhibition constant for AMP by up to 10-fold (6Liu F. Fromm H.J. J. Biol. Chem. 1988; 263: 9122-9128Abstract Full Text PDF PubMed Google Scholar). FBPase is a tetramer of identical subunits (Mr = 37,000). To a first approximation, these subunits lie in the same plane and occupy the corners of a square in one of the principal quaternary states of the mammalian enzyme (R-state). By past convention, subunit C1 occupies the upper left-hand corner, with subunits C2–C4 following in a clockwise direction. AMP causes a transition from the R-state to the T-state, driving a 17° rotation of the C1-C2 subunit pair with respect to the C3-C4 pair about a molecular 2-fold axis of symmetry (10Shyur 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). Complexes of FBPase with AMP in the presence of F16P2, F26P2, and fructose 6-phosphate are all in the T-state (9Zhang Y. Liang J.-Y. Huang S. Lipscomb W.N. J. Mol. Biol. 1994; 244: 609-624Crossref PubMed Scopus (93) Google Scholar, 11Ke H.M. Zhang Y.P. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5243-5247Crossref PubMed Scopus (119) Google Scholar, 12Villeret V. Huang S. Zhang Y. Lipscomb W.N. Biochemistry. 1995; 34: 4307-4315Crossref PubMed Scopus (51) Google Scholar), whereas in the absence of AMP, the enzyme has appeared in the R-state in crystal structures (13Choe J.-Y. Poland B.W. Fromm H.J. Honzatko R.B. 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