The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)nC>p, in which C>p indicates a 2′3′-cyclic phosphate. This is a confirmation of the proposed mechanism for Rnase A, wherein the so-called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (k7q/r10q-rnase A) lacking p2 (a phosphate binding subsite adjacent, on the 3′ side, to the main phosphate binding site P1) shows a clear exonucleolytic pattern; a mutant (k66q-rnase A) lacking P0 (a phosphate binding subsite adjacent, on the 5′ side, to the main phosphate binding site P1) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p2 mutant, the amide group of glutamine can form a hydrogen bond with the 2′,3′-cyclic terminal phosphate, whereas the distance to a 3′,5′-phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p2 mutant.
- Oligocytidylic acids
- Ribonuclease A