Enthalpic and entropic determinants for the specificity of alkylation of the histidine‐12 residue of ribonuclease A by four bromoacetamido nucleoside affinity labels and bromoacetamide

The binding and alkylation rate constants for the reaction of four bromoacetamido pyrimidine nucleosides and bromoacetamide with bovine pancreatic ribonuclease A (RNase A) have been determined as a function of temperature. The four nucleoside derivatives react exclusively or preferentially with the NE2 atom of histidine‐12 and include 2′‐bromoacetamido‐2′‐deoxyuridine, 2′‐bromoacetamido‐2′‐deoxyxylofuranosyl‐uracil, 3′‐bromoacetamido‐3′‐deoxythymidine and 3′‐bromoacetamido‐3′‐deoxyarabinofuranosyluracil. Transition‐state parameters, δH^‡ and AS^‡, reveal that nucleosides with “up” OH groups experience relative rate enhancements which have been attributed to contacts between these groups and the enzyme in the transition state (1). Variations in alkylation rates are explained in terms of different degrees of entropic destabilization (2) of the nucleosides in the enzyme affinity label complex.