Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: transition state and the C-terminal function

Proteins. 2012 Apr;80(4):1143-53. doi: 10.1002/prot.24015. Epub 2012 Jan 31.

Abstract

The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P(2)ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF(4)), and in a complex with pyrophosphate (PFKFB3•PP(i)) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3-P•F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF(4) and PFKFB3•PP(i) complexes were obtained by soaking. The PFKFB3•AlF(4) and PFKFB3•PP(i) complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ~5.1 Å, and the pivotal point 2-P is on the same line, suggesting an "in-line" transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3•PP(i), implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P(2) ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aluminum Compounds / chemistry
  • Amino Acid Sequence
  • Catalytic Domain*
  • Conserved Sequence
  • Diphosphates / chemistry
  • Enzyme Activation
  • Enzyme Assays
  • Enzyme Stability
  • Escherichia coli / chemistry
  • Fluorides / chemistry
  • Humans
  • Isoenzymes / chemistry*
  • Ligands
  • Models, Molecular
  • Molecular Sequence Data
  • Multiprotein Complexes / chemistry*
  • Phosphofructokinase-2 / chemistry*
  • Protein Binding
  • Protein Isoforms / chemistry
  • Proteolysis
  • Sequence Alignment
  • Structure-Activity Relationship
  • Water / chemistry

Substances

  • Aluminum Compounds
  • Diphosphates
  • Isoenzymes
  • Ligands
  • Multiprotein Complexes
  • Protein Isoforms
  • Water
  • tetrafluoroaluminate
  • diphosphoric acid
  • PFKFB3 protein, human
  • Phosphofructokinase-2
  • Fluorides