Crystal structures of fructose 1,6-bisphosphatase: Mechanism of catalysis and allosteric inhibition revealed in product complexes

Crystal structures of metal-product complexes of fructose 1,6-bisphosphatase (FBPase) reveal competition between AMP and divalent cations. In the presence of AMP, the Zn2+-product and Mg2+-product complexes have a divalent cation present only at one of three metal binding sites (site 1). The enzyme is in the T-state conformation with a disordered loop of residues 52-72 (loop 52-72). In the absence of AMP, the enzyme crystallizes in the R-state conformation, with loop 52-72 associated with the active site. In structures without AMP, three metal-binding sites are occupied by Zn2+ and two of three metal sites (sites 1 and 2) by Mg2+. Evidently, the association of AMP with FBPase disorders loop 52-72, the consequence of which is the release of cations from two of three metal binding sites. In the Mg2+ complexes (but not the Zn2+ complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site I and is oriented for a nucleophilic attack on the bound phosphate molecule. A mechanism is presented for the forward reaction, in which Asp74 and Glu98 together generate a hydroxide anion coordinated to the Mg2+ at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg2+ at Site 1.