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°, 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 and the 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, K_b, is 0.087 M and k₃ is 35.1 × 10^-4sec^-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 K_i 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.