Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 6-phosphate, AMP, and magnesium.

Ke, Hengming; Zhang, Yiping; Lipscomb, William N.

doi:10.1073/pnas.87.14.5243

Cited by 115 publications

(98 citation statements)

References 47 publications

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“…5; Kinemage 3). The syn conformation was first observed in the 2.5 A structure of the native enzyme complexed with Fru-6-P and AMP (Ke et al, 1990). This conformation was confirmed by inspection of the omit maps and analysis of the distribution of temperature factors of the AMP molecule.…”

Section: Allosteric Site and A M P Bindingmentioning

confidence: 57%

“…The phosphate group of the inhibitor is held by numerous hydrogen bonds to Tyr-215, Asn-212, Tyr-244, Tyr-264, Lys-274, 1994). and the 0 3 oxygen of the furanose ring to the amide nitrogen of Met-248 and carboxyl group of Asp-121 in the very same manner as in the wild-type enzyme (Ke et al, 1990). The absence of Arg-243 is compensated by additional hydrogen bonds to solvent molecules.…”

Section: Active Site and Binding Of Fru-6-pmentioning

confidence: 84%

“…Inhibition by AMP is cooperative, with a Hill coefficient near 2 (Taketa & Pogell, 1965;Nimmo & Tipton, 1975a), whereas Fru-2,6-P2 binds to the active sites of the enzyme (Ke et al, 1989a) in competition with the substrate Fru-1,6-P2 (Pilkis et al, 1981). The AMP binding site on each subunit is 28 A distant from the active site (Keet al, 1991a) and the metal-binding sites are located between the Fru-1,6-P, and AMP domains (Ke et al, 1990).…”

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confidence: 99%

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Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli

et al. 1996

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The active site of pig kidney fructose-l,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-l,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-l,6-bisphosphate was reduced IO-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chern 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243 + Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.

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Section: Allosteric Site and A M P Bindingmentioning

confidence: 57%

Section: Active Site and Binding Of Fru-6-pmentioning

confidence: 84%

mentioning

confidence: 99%

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Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli

et al. 1996

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“…AMP causes a transition from the R-state to the T-state, driving a 17°r otation of the C1-C2 subunit pair with respect to the C3-C4 pair about a molecular 2-fold axis of symmetry (10). Complexes of FBPase with AMP in the presence of F16P 2 , F26P 2 , and fructose 6-phosphate are all in the T-state (9,11,12), whereas in the absence of AMP, the enzyme has appeared in the R-state in crystal structures (13,14).…”

mentioning

confidence: 99%

The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Nelson

Kurbanov

Honzatko

et al. 2001

Journal of Biological Chemistry

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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 reduces k cat by 30-fold and Mg 2؉ affinity by 20-fold and eliminates cooperativity in Mg 2؉ 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 k cat and Mg 2؉ affinity significantly but has no effect on AMP inhibition. The mutation of Asp 9 to alanine reproduces the weakened affinity for Mg 2؉ observed in the deletion mutants, and the mutation of Ile 10 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 thioredoxinregulated FBPase from the chloroplast.Fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase 1 ) catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate and inorganic phosphate (P i ) (1-3). 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 (4). FBPase activity requires divalent cations such as Mg 2ϩ , Mn 2ϩ , or Zn 2ϩ , and plots of velocity versus metal ion concentration are sigmoidal with a Hill coefficient of 2.0 (5-7). AMP binds cooperatively (Hill coefficient of 2) 28 Å from the nearest active site (8) and inhibits the enzyme, whereas F26P 2 binds at the active site (9). Inhibition of FBPase by AMP and F26P 2 is synergistic. F26P 2 can lower the apparent inhibition constant for AMP by up to 10-fold (6).FBPase is a tetramer of identical subunits (M r ϭ 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°r otation of the C1-C2 subunit pair with respect to the C3-C4 pair about a molecular 2-fold axis of symmetry (10). Complexes of FBPase with AMP in the presence of F16P 2 , F26P 2 , and fructose 6-phosphate are all in the T-state (9, 11, 12), whereas in the absence of AMP, the enzyme has appeared in the R-state in crystal structures (13,14).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 ...

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“…The four identical subunits (C1, C2, C3, and C4) of FBPase each consist of single AMP (residues 1-200) and FBP (residues 200 -335) binding domains (10). The tetramer is roughly a square with the upper left vertex occupied by subunit C1 followed by C2, C3, and C4 in a clockwise sense.…”

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confidence: 99%

Major Changes in the Kinetic Mechanism of AMP Inhibition and AMP Cooperativity Attend the Mutation of Arg49 in Fructose-1,6-bisphosphatase

Shyur

Poland

Honzatko

et al. 1997

Journal of Biological Chemistry

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The significance of subunit interface residues Arg 49 and Lys 50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys 50 3 Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg 49 49 3 Cys mutant). In addition, AMP cooperativity was absent in the Arg 49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steadystate Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11, FBPase 1 ) governs a crucial step in carbohydrate metabolism, the hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P 2 ) to fructose 6-phosphate and inorganic phosphate (P i ) in the presence of required divalent cations such as Mg 2ϩ (1, 2). In mammals, FBPase is a homotetramer, inhibited competitively by fructose 2,6-bisphosphate (Fru-2,6-P2) (3-6) and allosterically by AMP (7-9). Crystallographic complexes reveal 1 AMP binding site/subunit (10) and a single common binding site for Fru-2,6-P 2 and fructose 6-phosphate approximately 20 Å away from the AMP site. Metal ions bind at or near the active site. Activation of FBPase by Mg 2ϩ exhibits sigmoidal kinetics with a Hill coefficient of 2 at neutral pH but exhibits hyperbolic kinetics at pH 9.6 (11, 12).In the context of FBPase kinetics, the binding of AMP results in a diverse and complex set of phenomena. The inhibition of FBPase by AMP and Fru-2,6-P 2 is synergistic (13, 14), and the Fru-2,6-P 2 -induced enhancement of AMP binding is attributed to a decrease in the k off for the nucleotide (4). AMP and Mg 2ϩ are mutually exclusive in their binding to FBPase (11,15). Crystallographic studies have shown that AMP indirectly perturbs metal binding sites (16). AMP inhibition is cooperative, with a Hill coefficient of 2 (7-9). The first two molecules of AMP putatively bind with positive cooperativity, whereas the last two molecules bind with negative cooperativity (17). The mechanism for AMP inhibition is nonlinear and noncompetitive, with respect to Fru-1,6-P 2 and nonlinear and competitive with respect to Mg 2ϩ (11).The four identical subunits (C1, C2, C3, and C4) of FBPase each consist of single AMP (residues 1-200) and FBP (residues 200 -335) binding domains (10). The tetramer is roughly a square with the upper left vertex occupied by subunit C1 followed by C2, C3, and C4 in a clockwise sense. Structures of AMP complexes of FBPase define the T-state, whereas structures of FBPase in complexes with substrates or substrate analogs without AMP define the R-state. To a first approximation, the...

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Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 6-phosphate, AMP, and magnesium.

Cited by 115 publications

References 47 publications

Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli

Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli

The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Major Changes in the Kinetic Mechanism of AMP Inhibition and AMP Cooperativity Attend the Mutation of Arg49 in Fructose-1,6-bisphosphatase

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