It is generally assumed that an enzyme-substrate (ES) complex contains all
components necessary for catalysis and that conversion to products occurs by
rearrangement of atoms, protons, and electrons. However, we find that DNA
synthesis does not occur in a fully assembled DNA polymerase-DNA-deoxynucleoside
triphosphate complex with two canonical metal ions bound. Using time-resolved
x-ray crystallography, we show that the phosphoryltransfer reaction takes place
only after the ES complex captures a third divalent cation that is not
coordinated by the enzyme. Binding of the third cation is incompatible with the
basal ES complex and requires thermal activation of the ES for entry. It is
likely that the third cation provides the ultimate boost over the energy barrier
to catalysis of DNA synthesis.
