Modification of bovine pancreatic ribonuclease A with 6-chloropurine riboside.
The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine riboside was studied to obtain information about the role of the purine nucleoside moiety of the ribonucleic acid in the enzyme-substrate interaction. The residues involved in the reaction were identified, after performic acid oxidation and trypsin digestion, by reverse-phase HPLC peptide mapping. The labeled peptides were detected by following the absorbance at 254 nm, and amino acid analyses of these peptides showed that the reaction had taken place with the amino groups of Lys-1, -37, -41, and -91. The specificity of the reaction was unaffected by changing the ligand:protein molar ratio. Partial separation of the reaction products was accomplished by means of chromatography on CM-Sepharose: four labeled fractions corresponding to mono- and bisubstituted derivatives were found. One of the monosubstituted fractions (fraction E) contained a homogeneous protein with the nucleoside bound to the alpha-amino group of Lys-1 whereas the other (fraction D) was a mixture of derivatives labeled in the epsilon-amino group of Lys-1, -37, -41, and -91. Kinetic studies of these two monosubstituted fractions were performed with cytidine 2',3'-phosphate and ribonucleic acid as substrates. These derivatives showed a noncompetitive inhibition-like behavior with respect to RNase A. Results support the existence of several RNase A regions with affinity for purine nucleosides.
Alonso
J
J
Nogués
M V
MV
Cuchillo
C M
CM
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011684:Purine Nucleosides / Q000378:metabolism / Q000494:pharmacology*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin
D010446:Peptide Fragments; D011684:Purine Nucleosides; C024008:6-chloropurine riboside; D012259:Ribonuclease, Pancreatic; D014357:Trypsin