Mechanism of hydrolysis of phosphate esters by the dimetal center of 5'-nucleotidase based on crystal structures

J Mol Biol. 2001 May 25;309(1):239-54. doi: 10.1006/jmbi.2001.4656.


5'-Nucleotidase belongs to a large superfamily of distantly related dinuclear metallophosphatases including the Ser/Thr protein phosphatases and purple acid phosphatases. The protein undergoes a 96 degrees domain rotation between an open (inactive) and a closed (active) enzyme form. Complex structures of the closed form with the products adenosine and phosphate, and with the substrate analogue inhibitor alpha,beta-methylene ADP, have been determined at 2.1 A and 1.85 A resolution, respectively. In addition, a complex of the open form of 5'-nucleotidase with ATP was analyzed at a resolution of 1.7 A. These structures show that the adenosine group binds to a specific binding pocket of the C-terminal domain. The adenine ring is stacked between Phe429 and Phe498. The N-terminal domain provides the ligands to the dimetal cluster and the conserved His117, which together form the catalytic core structure. However, the three C-terminal arginine residues 375, 379 and 410, which are involved in substrate binding, may also play a role in transition-state stabilization. The beta-phosphate group of the inhibitor is terminally coordinated to the site 2 metal ion. The site 1 metal ion coordinates a water molecule which is in an ideal position for a nucleophilic attack on the phosphorus atom, assuming an in-line mechanism of phosphoryl transfer. Another water molecule bridges the two metal ions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / antagonists & inhibitors
  • 5'-Nucleotidase / chemistry*
  • 5'-Nucleotidase / metabolism*
  • Acid Phosphatase / chemistry
  • Adenosine Diphosphate / analogs & derivatives*
  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Animals
  • Binding Sites
  • Catalysis
  • Conserved Sequence
  • Crystallography, X-Ray
  • Escherichia coli / enzymology*
  • Glycoproteins / chemistry
  • Hydrolysis
  • Ions
  • Ligands
  • Metals / metabolism
  • Models, Molecular
  • Phosphates / metabolism*
  • Phosphoprotein Phosphatases / chemistry
  • Protein Binding
  • Protein Conformation
  • Rotation
  • Static Electricity
  • Structure-Activity Relationship
  • Substrate Specificity
  • Temperature
  • Water / chemistry
  • Water / metabolism


  • Glycoproteins
  • Ions
  • Ligands
  • Metals
  • Phosphates
  • Water
  • alpha,beta-methyleneadenosine 5'-diphosphate
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • purple acid phosphatase
  • Phosphoprotein Phosphatases
  • Acid Phosphatase
  • 5'-Nucleotidase

Associated data

  • PDB/1HO5
  • PDB/1HP1
  • PDB/1HPU