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  • The EMBO Journal (2003) 22, 995 - 1004
  • doi:10.1093/emboj/cdg098

Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease

Mark R. Tock1, Elaine Frary1, Jon R. Sayers2 and Jane A. Grasby1

  1. Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK
  2. University of Sheffield Medical School, Division of Genomic Medicine, Krebs Institute, Sheffield S10 2RX, UK

Correspondence to:

Jane A. Grasby, E-mail: j.a.grasby@sheffield.ac.uk

Received 25 November 2002; Accepted 6 January 2003; Revised 2 January 2003

On the basis of structural work, metal ions are proposed to play a catalytic role in reactions mediated by many phosphoryl transfer enzymes. To gain dynamic support for such mechanisms, the role of metal ion cofactors in phosphate diester hydrolysis catalysed by a flap endonuclease has been studied. The pH maximal rate profiles were measured in the presence of various metal ion cofactors; in each case, a single ionic form of the enzyme/cofactor accounts for the pH dependence. The kinetic pKas display good correlation with the acidity of the corresponding hexahydrated metal ions, which strongly suggests a role for metal-bound hydroxide, or its equivalent ionic species, in the reaction. Comparing rates of reaction in the pH-independent regions, a small negative betanuc value is observed. This suggests that expected trends in the nucleophilicity of the various metal-bound hydroxides are balanced by a second form of metal ion catalysis that is related to the acidity of the hexahydrated metal ion. This is likely to be either electrophilic catalysis or leaving group activation.

  • Keywords:

    • metal ion catalysis,
    • nuclease,
    • pH dependency