Metal-binding and folding thermodynamics of Escherichia coli ribonuclease HI related to its catalytic function

Abstract

Escherichia coli ribonuclease HI (RNH) hydrolyzes the RNA strands of RNA/DNA hybrids in the presence of Mg²⁺ at the highest level, relative to other metal ions. The Mg²⁺ binding affinity was 8.39 × 10³ M^-1, which was lower than those of other metal ions. The low-affinity binder can express the maximum catalytic activity of RNH. The stability of RNH increased with increasing metal ion concentration, except for Zn²⁺. The thermodynamic origin for enhancing the stability of RNH with Mg²⁺ was more favorable entropy compared to those with other metal ions, indicating that Mg²⁺ binding changes the RNH structure while maintaining flexibility. Upon H124A mutation, the metal ion binding affinities decreased for Mn²⁺ and Zn²⁺ to a relatively large extent. The present thermodynamic analyses provide information on the structural dynamics of RNH with metal ion exchangeable binding, which can reasonably explain the metal-ion-dependent catalytic activity.

Keywords: Crystal structure; Differential scanning calorimetry; Enzyme; Isothermal titration calorimetry; Metal ion binding; Structural dynamics.