Ribonuclease A mutants His¹¹⁹ Asn: The role of histidine in catalysis

Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His¹², His¹¹⁹, and Lys⁴¹) are known to be involved in catalysis. Mutation of His¹¹⁹ to asparagine was generated to study the role of His¹¹⁹ in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His¹¹⁹ of the wild-type enzyme must be protonated in an enzyme-substrate productive complex. Thus our results indicate that a contribution of protonated His¹¹⁹ into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His¹¹⁹ participates directly in a transition state stabilization via hydrogen bonding.