1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A.
The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004720:Endonucleases / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007291:Inosine Monophosphate / Q000031:analogs & derivatives*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D011522:Protons; D011685:Purine Nucleotides / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000378:metabolism*
D009711:Nucleotides; D011522:Protons; D011685:Purine Nucleotides; D011743:Pyrimidines; D007291:Inosine Monophosphate; D006639:Histidine; C018488:6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate; D004720:Endonucleases; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic