Overview of RNase A Structure/Function Relationships
Ribonuclease A (RNase A) is a digestive enzyme secreted by the pancreas that specifically "digests" or hydrolyzes RNA (but not DNA) polymers by endonuclease cleaveage of the phosphodiester bonds forming the covalent links between adjacent ribonucleotide residues in these molecules.

The relatively small, 124 amino acid sequence of RNase A contains four His residues, two of which -- His12 and His119 are directly implicated as catalytic fresidues of this enzyme. The imidazole ring of His12 has an anonymously low pK value (pK < 6.0) suggesting it must be deprotonated for catalysis. Conversely, the imidazole ring of His119 has an anonymously high pK value (pK > 6.0) suggesting it must be protonated for catalysis.

Click here to view the microenvironments surround the of the 4 titratable HIS sidechains in RNase A and their corresponding NMR acid-base titration analysis.

A two-step reaction is proposed for hydrolysis of RNA.
  1. His12 acts as a general base, accepting the 2'-OH proton of the sessile ribonucleic sugar ring.  This promotes a nucleophilic attack by the 2'-O on the more positively-charged phosphorus (P) atom thereby creating a 2’,3’-cyclic ribonucleotide phosphate intermediate. The creation of this intermediate is facilitated by the imidazole of His119 which acts as a general acid, donating its proton to the oxygen atom in the susceptible P-O-R' bond.
  2. For the second step of the reaction, the general acid and base activities of the sidechains of these two His residue are reversed. His12 acts as a general acid, donating its newly acquired proton to the 2’,3’-cyclic ribonucleotide phosphate intermediate, while His119 which acts as a general base, accepting a proton from water to promote hydroxyl attack on the same 2’,3’-cyclic intermediate.
Uridine vanadate is a potent competitive inhibitor of RNase A. What makes it so potent is the fact that its structure mimics a transition state intermediate in the reaction. Specifically, the oxidized vanadium atom is pentavalent ion with five coordinated oxygen atoms whose stable structure is believed to mimic the bipyramid structure of an unstable pentavalent phosphodiester intermediate that transiently forms in the active site of the enzyme.
rnasase2.gif (33853 bytes)
Legend: 2',3'-cyclic uridine vanadate complex with RNase A active site:  The pentacovalently coordinated vanadium (V) atom of the inhibitor forms metal bonds with 5 oxygen atoms (red spheres) creating a bipyramidal 2',3' cyclic ribonucleotide (black wireframe) linked to uracil. Specific active site residue contacts with His 119, His12, Lys41, and Thr45 are shown with distance measurements indicated in Ångstroms.

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© Duane W. Sears
Revised: October 07, 2010