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

Biochemistry. 2000 Jul 25;39(29):8565-74. doi: 10.1021/bi000574g.

Abstract

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 Zn(2+)-product and Mg(2+)-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 Zn(2+) and two of three metal sites (sites 1 and 2) by Mg(2+). 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 Mg(2+) complexes (but not the Zn(2+) complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site 1 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 Mg(2+) at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg(2+) at site 1.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Allosteric Regulation
  • Allosteric Site
  • Animals
  • Catalysis
  • Crystallography, X-Ray
  • Fructose-Bisphosphatase / antagonists & inhibitors
  • Fructose-Bisphosphatase / chemistry*
  • Fructose-Bisphosphatase / metabolism*
  • In Vitro Techniques
  • Magnesium / chemistry
  • Models, Molecular
  • Protein Conformation
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Static Electricity
  • Swine
  • Zinc / chemistry

Substances

  • Recombinant Proteins
  • Fructose-Bisphosphatase
  • Magnesium
  • Zinc

Associated data

  • PDB/1CNQ
  • PDB/1EYI
  • PDB/1EYJ
  • PDB/1EYK