Role of a Dynamic Loop in Cation Activation and Allosteric Regulation of Recombinant Porcine Fructose-1,6-bisphosphatase^†,‡

A disordered loop (loop 52−72, residues 52−72) in crystal structures of fructose-1,6-bisphosphatase (FBPase) has been implicated in regulatory and catalytic phenomena by studies in directed mutation. A crystal structure of FBPase in a complex with three zinc cations and the products fructose 6-phosphate (F6P) and phosphate (P_i) reveals loop 52−72 for the first time in a well-defined conformation with strong electron density. Loop 52−57 interacts primarily with the active site of its own subunit. Asp68 of the loop hydrogen bonds with Arg276 and a zinc cation located at the putative potassium activation site. Leu56 and Tyr57 of the loop pack against hydrophobic residues from two separate subunits of FBPase. A mechanism of allosteric regulation of catalysis is presented, in which AMP, by binding to its allosteric pocket, displaces loop 52−72 from the active site. Furthermore, the current structure suggests that both the α- and β-anomers of F6P can be substrates in the reverse reaction catalyzed by FBPase. Mechanisms of catalysis are proposed for the reverse reaction in which Asp121 serves as a catalytic base for the α-anomer and Glu280 serves as a catalytic base for the β-anomer.