Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P [RNA]

March 27th, 2015 by Howard, M. J., Klemm, B. P., Fierke, C. A.

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single-turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least 2 active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified 2 aspartates important for enhancing metal ion affinity. The single-turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ~8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P [RNA]