Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease

Biochemistry. 1999 May 18;38(20):6576-86. doi: 10.1021/bi9901580.


The rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.

Publication types

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

MeSH terms

  • Alanine / genetics
  • Binding Sites / genetics
  • Catalysis
  • Cations, Divalent / chemistry
  • Cations, Divalent / metabolism
  • Deoxyribonucleases, Type II Site-Specific / chemistry*
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Energy Transfer
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Hydrogen-Ion Concentration
  • Kinetics
  • Metals / chemistry*
  • Metals / metabolism
  • Mutagenesis, Site-Directed
  • Phosphates / chemistry*
  • Phosphates / metabolism
  • Protein Conformation
  • Structure-Activity Relationship
  • Thermodynamics
  • Threonine / genetics


  • Cations, Divalent
  • Metals
  • Phosphates
  • Threonine
  • Deoxyribonucleases, Type II Site-Specific
  • GATATC-specific type II deoxyribonucleases
  • Alanine