Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis.

Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while Mg and Mg have similar binding constants, the dissociation rate of Mg is two order of magnitude lower than that of Mg due to a kinetic-barrier-difference.