A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp 187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R-and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52؊72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg 2؉, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) 1 catalyzes a tightly regulated step of gluconeogenesis, the hydrolysis of fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate (F6P) and P i (1, 2). AMP and F26P 2 (binding to allosteric and active sites, respectively) inhibit FBPase, while simultaneously activating its counterpart in glycolysis, fructose-6-phosphate 1-kinase (3, 4). Biosynthesis and degradation of F26P 2 is subject to hormonal control principally by glucagon and insulin (4, 5 FBPase is a homotetramer (subunit M r of 37,000 (11)) and exists in at least two distinct quaternary conformations called R and T (12Ϫ14). AMP induces the transition from the active R-state to the inactive (or less active) T-state. Substrates or products in combination with metal cations stabilize the Rstate conformation. A proposed mechanism for allosteric regulation of catalysis involves three conformational states of loop 52Ϫ72 called engaged, disengaged, and disordered (15). AMP alone or with F26P 2 stabilizes a disengaged loop (16, 17), whereas metals with products stabilize an engaged loop (10, 17Ϫ19). In active forms of the enzyme, loop 52Ϫ72 probably cycles between its engaged and disordered conformations (15,18). 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-st...

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