Dynamic coordination of two-metal-ions & chemo-mechanical catalysis by an exonuclease
  • Gwangrog Lee Ph.D
  • SLS Colloquia / Sep 26th 4pm / bldg.110 room N104
Abstract

First part: Metal ions at the active site of many enzymes act as essential cofactors, and their dynamic fluctuations, if any, could influence enzyme activity. We use λ-exonuclease as a model enzyme with two Mg2+ binding sites and investigate activity at various concentrations of Mg2+ by single-molecule-FRET. We find that while MgA2+ and MgB2+ exhibit similar binding constants, the dissociation rate of MgA2+ is two order of magnitude lower than that of MgB2+ due to a kinetic-barrier-difference of ~5.2 . At physiological 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 with enzyme-translocation. Second part: Phosphates along DNA function as chemical energy for a nucleic acid motor to drive enzymatic translocation. We discovered that 5' phosphates, generated at each cleavage step of the reaction, chemo-mechanically facilitate the translocation and subsequent post-cleavage melting of the terminal base pairs. We also found that transient coupling between the enzyme and DNA substrate significantly alters the translocation by chemical friction between a key reside of the protein (ARG45) and electrostatic potential (EP) along the minor groove of DNA. The fiction provides attractive coupling between DNA and the protein through chemical ratcheting for unidirectional translocation. Our study provides new insights into 1) the importance of metal-ion-coordination dynamics during the enzymatic reaction, 2) the versatile roles of phosphates and 3) a role of the minor groove in regulating enzymatic activity via chemical friction.