Abstract
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 Mg2+ binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while MgA2+ and MgB2+ have similar binding constants, the dissociation rate of MgA2+ is two order of magnitude lower than that of MgB2+ due to a kinetic-barrier-difference. At physiological Mg2+ concentration, the MgB2+ ion near the 5’-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of MgA2+ result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.
Highlights
Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity
We perform kinetic modeling of Mg2+-dependent degradation and molecular dynamics (MD) simulations to correlate the metal-ion chemistry and enzyme activity and to infer a molecular mechanism that can best explain the diverse patterns observed in the fluorescence resonance energy transfer (FRET) time trajectories
We find that the two metal ions in the active site (MgA2+ and MgB2+) have similar binding constants, but asymmetric thermodynamic stabilities during the enzymatic reaction
Summary
Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. We use λ-exonuclease as a model enzyme with two Mg2+ binding sites and probe activity at various concentrations of magnesium by singlemolecule-FRET. We use λ-exonuclease as a model system to investigate the microscopic underpinnings of how the two catalytic metal ions promote catalysis and address how their dynamics affect overall enzymatic activity. We perform kinetic modeling of Mg2+-dependent degradation and molecular dynamics (MD) simulations to correlate the metal-ion chemistry and enzyme activity (including cleavage and translocation) and to infer a molecular mechanism that can best explain the diverse patterns observed in the FRET time trajectories. We find that the two metal ions in the active site (MgA2+ and MgB2+) have similar binding constants, but asymmetric thermodynamic stabilities during the enzymatic reaction
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