Identification of the ternary complex of ribonuclease HI:RNA/DNA hybrid:metal ions by ESI mass spectrometry

Abstract

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme-substrate complex in the presence of divalent metal ions (Mn²⁺ or Zn²⁺) using electrospray ionization-mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn²⁺ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn²⁺ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn²⁺], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn²⁺ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate.

Keywords: manganese; mass spectrometry (MS); metal ion–protein interaction; ribonuclease; zinc.