Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease.
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.
Tock
Mark R
MR
Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
Frary
Elaine
E
Sayers
Jon R
JR
Grasby
Jane A
JA
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
D008670:Metals; D003035:Cobalt; D008345:Manganese; D005090:Exodeoxyribonucleases; C079044:T5 D15 exonuclease; D008274:Magnesium