The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size.
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)(n)C>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 p(2) (a phosphate binding subsite adjacent, on the 3' side, to the main phosphate binding site p(1)) shows a clear exonucleolytic pattern; a mutant (K66Q-RNase A) lacking p(0) (a phosphate binding subsite adjacent, on the 5' side, to the main phosphate binding site p(1)) 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 p(2) 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 p(2) mutant.
Cuchillo
Claudi M
CM
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Moussaoui
Mohamed
M
Barman
Tom
T
Travers
Franck
F
Nogués
M Victòria
MV
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009841:Oligonucleotides / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors
D009841:Oligonucleotides; D012259:Ribonuclease, Pancreatic