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

Abstract

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...

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

“…Nonetheless, the geometric relationship between P i and F6P ( Fig. 2A) is consistent with an associative reaction mechanism, the details of which have been presented elsewhere (40); Asp 74 (the pK a of which is raised by the proximity of Glu 98 ) abstracts a proton from a water molecule coordinated to the Mg 2ϩ at site 2 or site 3. The resulting Mg 2ϩ -coordinated hydroxide anion in turn abstracts the proton from a second water molecule (the attacking nucleophile) that bridges the magnesium cations at sites 2 and 3.…”

“…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.…”

“…As in the high K ϩ complex, the 1-OH group of F6P is in its displaced orientation. The loss of Glu 97 as a coordinating ligand to Mg 2ϩ at site 3 is linked perhaps to the binding of magnesium cations at sites (40). Glu 98 coordinates to Mg 2ϩ at site 4, whereas Asp 68 coordinates to Mg 2ϩ at site 3, but the binding of metal cations to sites 3 and 4 are probably mutually exclusive, as they are only 2.5 Å apart.…”

“…The Mg 2ϩ at site 4, a cationbinding site identified in Mg 2ϩ /Tl ϩ complexes of FBPase (40), may in fact play a direct role in catalysis (Fig. 3).…”

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

“…Fluorescence from a tryptophan reporter group at position 57 is consistent with the conformational states for loop 52Ϫ72, observed in crystal structures (20,21). Presumably, the engaged, disengaged, and disordered conformations of loop 52Ϫ72 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 (17,18,22,23).A recent report (24) in the literature identifies a new inhibitor site on FBPase, distinct from the active and the AMPbinding sites. The new family of anilinoquinazoline inhibitors was discovered by a search for potential drugs in the treatment of non-insulin-dependent diabetes.…”