The kinetics and specificity of the reaction of 2'(3')-O-bromoacetyluridine with bovine pancreatic ribonuclease A.
2'(3')-O-Bromoacetyluridine reacts rapidly and selectively with bovine pancreatic ribonuclease A at pH 5.5 and 25 degrees. Under conditions of high molar ratios of nucleoside derivative to enzyme, the only derivative is N-3-carboxymethylhistidine-12 ribonuclease A. The reaction occurs almost exclusively with the histidine-12 residue at the active site inactivation of the enzyme is accompanied by the stoichiometric disappearance of unmodified ribonuclease A and appearance of the product, N-3-carboxymethylhistidine-12 ribonuclease A. Kinetic studies indicate a mechanism involving saturation of the enzyme by the nucleoside derivative. The inhibitor constant, Kb, is 0.087 M and k3 is 35.1 times 10(-4) sec minus 1. The reaction of 2'(3')-O-bromoacetyluridine with the enzyme occurs at a rate approximately 3100 times greater than that corresponding to the reaction with L-histidine. The alkylation reaction is inhibited competitively by uridine with a Ki of 0.013 M. 2'(3')-O-Bromoacetyluridine inactivates ribonuclease A 4.5 times faster than bromoacetic acid and the specificity for alkylation of active-site histidine residues is different. 2'(3')-O-Bromoacetyluridine reacts 1000 times more rapidly with ribonuclease A than iodoacetamide. The contribution of nucleoside binding to the overall rate of alkylation is discussed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002852:Chromatography, Ion Exchange; D006639:Histidine / Q000032:analysis; D007460:Iodoacetamide; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D012260:Ribonucleases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014529:Uridine / Q000031:analogs & derivatives*
D000596:Amino Acids; D010446:Peptide Fragments; D006639:Histidine; D012260:Ribonucleases; D014529:Uridine; D007460:Iodoacetamide