Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate

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Abstract
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 K3 have been determined for each derivative. They are the following: 3''-(bromoacetamido)-3''-deoxyuridine, Kb = 0.062 M, k3 = 3.3 .times. 10-4 s-1; 2''-(bromoacetamido)-2''-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 .times. 10-4 s-1; 3''-(bromoacetamido)-3''-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 .times. 10-4 s-1; and 3''-(bromoacetamido)-3''-deoxythymidine, Kb = 0.094 M, k3 = 2.7 .times. 10-4 s-1. 3''-(Bromoacetamido)-3''-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2''-(brooacetamido)-2''-deoxyxylofuranosyluracil 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-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 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.