A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

Mohammed Moussaoui, Claudi M. Cuchillo, M. Victòria Nogués

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)


A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3′-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.
Original languageEnglish
Pages (from-to)99-109
JournalProtein Science
Publication statusPublished - 8 Jan 2007


  • Active site
  • Artificial enzymes
  • Catalytic mechanism; protein engineering
  • Enzyme kinetics
  • Ribonuclease A


Dive into the research topics of 'A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site'. Together they form a unique fingerprint.

Cite this