Reaction of (Bromoacetamido)nucleoside Affinity Labels with Ribonuclease A: Evidence for Steric Control of Reaction Specificity and Alkylation Rate

Charles F. Hummel, Matthew R. Pincus, Paul W. Brandt-Rauf, Gill M. Frei, Robert P. Carty

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


: Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3have been determined for each derivative. They are the following: 3/-(bromoacet-amido)-3/-deoxyuridine, Kb = 0.062 M, k3= 3.3 X 10–4s-1; 2‘-(bromoacetamido)-2’-deoxyxylofuranosyluracil, Kb= 0.18 M, k3 = 1700 X 10-4 s-1; 3‘-(bromoacetamido)-3’-deoxyarabinofuranosyluracil, Kb= 0.038 M, k3= 6.6 X 10-4 s-1; and 3/-(bromoacetamido)-3/-deoxythymidine, Kb = 0.094 M, k3= 2.7 X 10-4 s-1. 3‘-(Bromoacetamido)-3’-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-l, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-l and N-3. Both 2’-(bromoacetamido)-2/-deoxyxylo-furanosyluracil and 3/-(bromoacetamido)-3/-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3/-(Bromoacetamido)-3/-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-l of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-l of histidine-119. A specific interaction of the “down” 2’-OH group, unique to 3/-(bromoacetamido)-3/-deoxyuridine, serves to orient the 3/-bromoacetamido residue close to the imidazole ring of histidine-119. The 2’-OH group of 3/, 5/-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.

Original languageEnglish
Pages (from-to)135-146
Number of pages12
Issue number1
StatePublished - 1987
Externally publishedYes


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