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

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 X 10(-4) s-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-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'-(bromoacetamido)-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.

Ribonuclease A

“…150,151 The structure of the crystalline product of the alkylation of RNase A by 4 is known. 152 2′-(3′)-O-Bromoacetyluridine 214,215 and its amide analogues 3′-(bromoacetamido)-3′-deoxythymidine (5), 3′-(bromoacetamido)-3′-deoxyuridine, 3′-(bromoacetamido)-3′-deoxyarabinofuranosyluracil, 2′-(bromoacetamido)-2′-deoxyuridine, and 2′-(bromoacetamido)-2′-deoxyxylofuranosyluracil [216][217][218] alkylate the side chains of His12 or His119. The structures of the crystalline products of the alkylation of RNase A by 5 and by 3′-(bromoacetamido)-3′-deoxyuridine are known.…”

“…6-Chloropurine 9-β-D-ribofuranosyl 5‘-monophosphate ( 4 ) alkylates the α-amino group of Lys1, presumably after binding to the B3 subsite (Figure ). , The structure of the crystalline product of the alkylation of RNase A by 4 is known − alkylate the side chains of His12 or His119. The structures of the crystalline products of the alkylation of RNase A by 5 and by 3‘-(bromoacetamido)-3‘-deoxyuridine are known …”

Crystal structure of two covalent nucleoside derivatives of ribonuclease A

“…These two histidine residues, together with Lys41, form the catalytic site of RNase A. This study continues the already large body of crystallographic studies of this enzyme and of its complexes with various inhibitors, performed in order to understand the details of the mechanism of reaction catalyzed by RNase A [see review by Wlodawer (1985) and references cited therein; Campbell & Petsko, 1987; Wlodawer et al, 1988],RNase A reacts rapidly with a variety of bromoacetamido pyrimidine nucleosides to form covalent derivatives in which either Hisl2 or Hisl 19 is alkylated (Hummel et al, 1987).3'-(Bromoacetamido)-3,-deoxythymidine forms four deriva-•This research was sponsored in part by the National Cancer Institute, DHHS, under Contract N01-C0-74101 with BRI.…”

“…RNase A reacts rapidly with a variety of bromoacetamido pyrimidine nucleosides to form covalent derivatives in which either Hisl2 or Hisl 19 is alkylated (Hummel et al, 1987).…”

Utilization of the free energy of the reversible binding of protein and modifying agent towards the rate-enhancement of protein covalent modification

“…highly -: negative, activation entropy, are better suited as prospective affinity-based protein-modifying agents, since the rate-enhancement, and hence the selectivity of labelling, to be obtained with these agents will be quite high. High rate-enhancement factors when protein modification reactions are compared with model-compound reactions have been observed in the case of the alkylation of creatine phosphokinase by iodoacetamide (65-fold rate-enhancement; Jones et al, 1975), the alkylation of RNAase by bromoacetamidopyrimidine nucleosides (9100-fold and 400-fold rateenhancement; Hummel et al, 1987) and the irreversible inhibition of sulphate transport in human erythrocytes by an isothiocyanic acid ester (1045-fold rate enhancement; Rakitzis, 1987). However, it is not clear whether, in these cases, rate-enhancement of protein modification is due to the utilization of binding energy or to the use of a different reaction pathway.…”