Ribonuclease A: Structure, Function and Mechanism

Structure

Ribonuclease A (RNase A) was first crystallized over 80 years ago, and these crystals were shown to diffract to a resolution of 2 Å. RNase A was the first enzyme and third protein (after insulin and hemoglobin) for which a correct amino acid sequence was determined, and the third enzyme and fourth protein (after myoglobin, lysozyme, and carboxypeptidase A) whose three‑dimensional structure was determined by X‑ray diffraction analysis.

Ribonuclease A is small. The mature enzyme, as secreted by exocrine cells of the bovine pancreas, has only 124 amino acid residues. RNase A contains 19 of the 20 natural amino acids, lacking only tryptophan. The molecular formula of the native, uncharged enzyme is C₅₇₅H₉₀₇N₁₇₁O₁₉₂S₁₂. This formula corresponds to a molecular mass of 13686 Da. As a small protein, RNase A became a target of synthetic chemists and was the first protein to succumb to total synthesis. This preparation had low, but measurable, ribonucleolytic activity.

The overall shape of the enzyme resembles that of a kidney, with the active‑site residues lying in the cleft. The predominant elements of secondary structure are a long four‑stranded antiparallel β‑sheet and three short α‑helixes. The enzyme is cross‑linked by four disulfide bonds, which involve all eight of its cysteine residues. The peptide bonds preceding two of the four proline residues are in the cis (or E) conformation. These proline residues are in type VI reverse turns at opposite ends of the native enzyme.

Function

Ribonuclease A is used in the research lab and DNA extraction. It cleaves the cellular RNA (all types) which are not required for cells. It especially cleaves the single-stranded cellular RNAse. 

It is also used in plasmid and genomic RNA isolation and preparation, mutation mapping, reparation of RNA from recombinant proteins and nuclease assays. 

Mechanism

Ribonuclease A catalyzes the cleavage of the P‑O⁵′ bond of RNA. In one possible mechanism, the side chain of His12 acts as a base that abstracts a proton from the 2′‑oxygen of a substrate molecule, and thereby facilitates its attack on the phosphorus atom. This attack proceeds inline to displace a nucleoside. The side chain of His119 acts as an acid that protonates the 5′‑oxygen to facilitate its displacement. Both products are released to solvent. The slow hydrolysis of the nucleoside 2′,3′‑cyclic phosphodiester occurs in a separate process, and resembles the reverse of transphosphorylation. The side chain of Lys41 and the main chain of Phe120 enhance catalysis by stabilizing this transition state.

Reference

[1] Raines, R. T. (1998). Ribonuclease A. Chemical Reviews, 98 3, 1045–1066. https://doi.org/10.1021/cr960427h