title: Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease.
abstract: 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 pK(a)s 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 beta(nuc) 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.
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Tock
authors.forename: Elaine
authors.initials: E
authors.lastname: Frary
authors.forename: Jon R
authors.initials: JR
authors.lastname: Sayers
authors.forename: Jane A
authors.initials: JA
authors.lastname: Grasby
mesh_terms: D002384:Catalysis; D003035:Cobalt / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D008670:Metals; D003035:Cobalt; D008345:Manganese; D005090:Exodeoxyribonucleases; C079044:T5 D15 exonuclease; D008274:Magnesium