Glutamic Acid Residue 98 Is Critical for Catalysis in Pig Kidney Fructose-1,6-bisphosphatase

et al. 2003

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...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Choe

Iancu

et al. 2003

“…Glu 98 is a proton acceptor in hydrogen bonds with both of these water molecules. Glu 98 is required for catalysis; mutations at position 98 cause a 10,000-fold decrease in activity under conditions that support full activity of the wild-type enzyme (43). Asp 74 , another residue essential for catalysis (24), hydrogen bonds only with the water molecule coordinated to Mg 2ϩ at site 2.…”

Section: Discussionmentioning

confidence: 99%

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

Choe

Nelson

et al. 2003

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...

“…Mutations of Arg 49 and Lys 50 that result in significant functional perturbations are due most likely to their influence on the conformational dynamics of loop 52-72. Mutations of Asn 64 , Asp 74 , and Glu 98 , residues in or near loop 52-72, eliminate AMP cooperativity (43,44). The introduction of proline at position 50 dramatically alters the mechanism of AMP inhibition and, on the basis of fluorescence spectroscopy, greatly changes the conformational dynamics of loop 52-72 (45).…”

Section: Expression and Purification Of Wild-type And Mutantmentioning

confidence: 99%

Hybrid Tetramers of Porcine Liver Fructose-1,6-bisphosphatase Reveal Multiple Pathways of Allosteric Inhibition

Nelson

Honzatko

2002

Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R-to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R-to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.