Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance

Enzymatic digestion with a type IIP restriction endonuclease EcoRV was investigated on a DNA-immobilized 27-MHz quartz crystal microbalance (QCM). Real-time observations of both the enzyme binding process and the DNA cleavage process of EcoRV were followed by frequency (mass) changes on the QCM, which were dependent on divalent cations such as Ca²⁺ or Mg²⁺. In the presence of Ca²⁺, the site-specific binding of EcoRV to DNA could be observed, without the catalytic process. On the other hand, in the presence of Mg²⁺, both the binding of the enzyme to the specific DNA (mass increase) and the site-specific cleavage reaction (mass decrease) could be observed continuously from QCM frequency changes. From time courses of frequency (mass) changes, each kinetic parameter, namely binding rate constants (k_on), dissociation rate constants (k_off), dissociation constants (K_d) of EcoRV to DNA, and catalytic rate constant (k_cat) of the cleavage reaction, could be determined. The binding kinetic parameters of EcoRV in the presence of Ca²⁺ were consistent with those of the binding process followed by the cleavage process in the presence of Mg²⁺. The k_cat value obtained by the QCM method was also consistent with that obtained by other methods. This study is the first to simultaneously determine k_on, k_off, and k_cat for a type IIP restriction endonuclease on one device.