Crystal Structures of Fructose 1,6-Bisphosphatase:  Mechanism of Catalysis and Allosteric Inhibition Revealed in Product Complexes<sup>,</sup>

Choe, Juhui; Fromm, Herbert J.; Honzatko, Richard B.

doi:10.1021/bi000574g

Cited by 72 publications

(135 citation statements)

References 33 publications

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“…Control Complex (PDB: 1NUY)-Aside from its substantially higher resolution (Table I), the control complex is essentially identical to the Mg 2ϩ product complex of Choe et al (29,30,40). Orthophosphate is clearly at the active site, and Mg 2ϩ occupies sites 1, 2, and 3 (Fig.…”

Section: Resultsmentioning

confidence: 73%

“…The inclusion of 0.2 mM EDTA in the crystallization solution eliminates the anomalous signal at metal site 1. Furthermore, the tetrahedral coordination of the site-1 cation and the displaced conformation of the 1-OH group of F6P are characteristics of previously determined Zn 2ϩ -product complexes of FBPase (29,30). A mixture of Zn 2ϩ and Mg 2ϩ at occupancies of 0.25 and 0.75, respectively, account for the magnitude of the anomalous signal, and provide thermal parameters of ϳ24 Å 2 , equivalent to that of the site-1 Mg 2ϩ in the control structure The thermal parameter associated with the Mg 2ϩ at site 3 is higher than that of its counterpart in the control complex (30 versus 26 Å 2 ).…”

Section: Resultsmentioning

confidence: 80%

“…All complexes have the dynamic loop (residues 52Ϫ72) in its engaged conformation, as determined by the location of Tyr 57 (29,30). Conditions that result in the formation of metaphosphate differ only in pH (7 versus 9.6) or the concentration of K ϩ (10 versus 200 mM).…”

Section: Resultsmentioning

confidence: 99%

“…In past crystal structures of FBPase, only orthophosphate and F6P have appeared in the active site (29,30). Presumably, the observed complexes represent a minimum free energy under the conditions of the crystallization experiment.…”

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confidence: 91%

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Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Choe

Iancu

Fromm

et al. 2003

Journal of Biological Chemistry

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The hydrolysis of a phosphate ester can proceed through an intermediate of metaphosphate (dissociative mechanism) or through a trigonal bipryamidal transition state (associative mechanism). Model systems in solution support the dissociative pathway, whereas most enzymologists favor an associative mechanism for enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 Å). At neutral pH, products of the reaction (orthophosphate and fructose 6-phosphate) bind to the active site in a manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K ؉ (200 mM), or at pH 9.6, metaphosphate and water (or OH ؊ ) are in equilibrium with orthophosphate. Furthermore, one of the magnesium cations in the pH 9.6 complex resides in an alternative position, and suggests the possibility of metal cation migration as the 1-phosphoryl group of the substrate undergoes hydrolysis. To the best of our knowledge, the crystal structures reported here represent the first direct observation of metaphosphate in a condensed phase and may provide the structural basis for fundamental changes in the catalytic mechanism of fructose-1,6-bisphosphatase in response to pH and different metal cation activators.Phosphatases, mutases, kinases, and nucleases all catalyze phosphoryl transfer reactions central to biochemical processes that sustain life. The transfer of the phosphoryl group of a phosphate ester to water in most cases requires metal ions as cofactors, and proceeds either by way of a trigonal-bipyramidal transition state (associative mechanism), or through the formation of an unstable intermediate of metaphosphate (dissociative mechanism) (1Ϫ6). The metaphosphate anion (PO 3 Ϫ ) was first proposed as an intermediate in the hydrolysis of phosphate esters nearly 50 years ago (7,8). Although it exists as a stable entity in the gas phase, where it is relatively non-reactive (9), metaphosphate is unstable in aqueous solutions, and its existence is inferred only by indirect evidence (9Ϫ15). The likelihood of trapping metaphosphate in the active site of an enzyme is remote, because of the proximity of acceptor molecules in the active site. Yet an enzyme offers an advantage in that it reduces the free energy of the transition state. Hence, the active site itself could serve as a thermodynamic trap if metaphosphate, once generated, is denied access to an acceptor.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11, hereafter FBPase) 1 is a key regulatory enzyme in gluconeogenesis. FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate (F6P) and orthophosphate (P i ). The inhibition of FBPase in mammals results in reduced levels of serum glucose in the fasting state. Hence, FBPase is a target for the development of drugs in the treatment of non-insulin dependent diabetes, which afflicts over 15 million people in the United State...

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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

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confidence: 91%

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Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Choe

Iancu

Fromm

et al. 2003

Journal of Biological Chemistry

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“…It can be in at least three conformational states: engaged, disordered, and disengaged (17,20,23). The engaged conformation is arguably required for the high affinity association of divalent cations and in stabilizing the transition state (17,(23)(24)(25)(26).…”

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confidence: 99%

Interaction of Tl+ with Product Complexes of Fructose-1,6-bisphosphatase

Choe

Nelson

Fromm

et al. 2003

Journal of Biological Chemistry

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Fructose-1,6-bisphosphatase requires divalent cations (Mg 2؉ , Mn 2؉ , or Zn 2؉ ) for catalysis, but a diverse set of monovalent cations (K ؉ , Tl ؉ , Rb ؉ , or NH 4 ؉ ) will further enhance enzyme activity. Here, the interaction of Tl ؉ with fructose-1,6-bisphosphatase is explored under conditions that support catalysis. On the basis of initial velocity kinetics, Tl ؉ enhances catalysis by 20% with a K a of 1.3 mM and a Hill coefficient near unity. Crystal structures of enzyme complexes with Mg 2؉ , Tl ؉ , and reaction products, in which the concentration of Tl ؉ is 1 mM or less, reveal Mg 2؉ at metal sites 1, 2, and 3 of the active site, but little or no bound Tl ؉ . Intermediate concentrations of Tl ؉ (5؊20 mM) displace Mg 2؉ from site 3 and the 1-OH group of fructose 6-phosphate from in-line geometry with respect to bound orthophosphate. Loop 52؊72 appears in a new conformational state, differing from its engaged conformation by disorder in residues 61؊69. Tl ؉ does not bind to metal sites 1 or 2 in the presence of Mg 2؉ , but does bind to four other sites with partial occupancy. Two of four Tl ؉ sites probably represent alternative binding sites for the site 3 catalytic Mg 2؉ , whereas the other sites could play roles in monovalent cation activation.Fructose-1,6-bisphosphatase (FBPase, 1 EC 3.1.3.11) hydrolyzes fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate (F6P) and phosphate (P i ) (1-6). Fructose 2,6-bisphosphate (F26P 2 ) and AMP synergistically inhibit FBPase. AMP inhibits by way of an allosteric and cooperative mechanism with a Hill coefficient of 2 (7-9). F26P 2 competes with F16P 2 for the active site (10 -12). Coordinated regulation of glycolysis and gluconeogenesis occurs in vivo, largely because of opposite effects caused by F26P 2 on FBPase (inhibition) and fructose 6-phosphate 1-kinase (activation). Divalent cations (Mg 2ϩ , Mn 2ϩ , and/or Zn 2ϩ ) are an absolute requirement for FBPase activity. Enzyme activity increases sigmoidally as a function of divalent cation concentration at pH 7.5 (Hill coefficient of ϳ2), but at pH 9.6 the variation is hyperbolic (8,14).The mammalian enzyme is a homotetramer and exists in distinct conformational states, depending on the relative concentrations of active site ligands and AMP (15). With or without metal cofactors and/or other active site ligands, but in the absence of AMP, FBPase is in its R-state (16,17). In the presence of AMP, however, the top pair of subunits rotates 17°r elative to the bottom pair, resulting in the T-state conformer (18 -20). The minimum distance separating AMP molecules from any given active site is ϳ28 Å (18 -20). Yet studies in kinetics, NMR, fluorescence, and x-ray crystallography reveal competition between AMP and divalent cations (11, 20 -22).Loop 52Ϫ72 evidently plays a central role in allosteric inhibition of catalysis by AMP. It can be in at least three conformational states: engaged, disordered, and disengaged (17,20,23). The engaged conformation is arguably required for the high affinity association of divale...

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Is allostery a fuzzy concept?

Morea,

Angelucci,

Bellelli

2024

FEBS Open Bio

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Allostery is an important property of biological macromolecules which regulates diverse biological functions such as catalysis, signal transduction, transport, and molecular recognition. However, the concept was expressed using two different definitions by J. Monod and, over time, more have been added by different authors, making it fuzzy. Here, we reviewed the different meanings of allostery in the current literature and found that it has been used to indicate that the function of a protein is regulated by heterotropic ligands, and/or that the binding of ligands and substrates presents homotropic positive or negative cooperativity, whatever the hypothesized or demonstrated reaction mechanism might be. Thus, proteins defined to be allosteric include not only those that obey the two‐state concerted model, but also those that obey different reaction mechanisms such as ligand‐induced fit, possibly coupled to sequential structure changes, and ligand‐linked dissociation‐association. Since each reaction mechanism requires its own mathematical description and is defined by it, there are many possible ‘allosteries’. This lack of clarity is made even fuzzier by the fact that the reaction mechanism is often assigned imprecisely and/or implicitly in the absence of the necessary experimental evidence. In this review, we examine a list of proteins that have been defined to be allosteric and attempt to assign a reaction mechanism to as many as possible.

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Crystal Structures of Fructose 1,6-Bisphosphatase: Mechanism of Catalysis and Allosteric Inhibition Revealed in Product Complexes^,

Cited by 72 publications

References 33 publications

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Interaction of Tl+ with Product Complexes of Fructose-1,6-bisphosphatase

Is allostery a fuzzy concept?

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Crystal Structures of Fructose 1,6-Bisphosphatase: Mechanism of Catalysis and Allosteric Inhibition Revealed in Product Complexes,

Cited by 72 publications

References 33 publications

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Interaction of Tl+ with Product Complexes of Fructose-1,6-bisphosphatase

Is allostery a fuzzy concept?

Contact Info

Product

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About

Crystal Structures of Fructose 1,6-Bisphosphatase: Mechanism of Catalysis and Allosteric Inhibition Revealed in Product Complexes^,