Hummel C F, Pincus M R, Brandt-Rauf P W, Frei G M, Carty R P
Biochemistry. 1987 Jan 13;26(1):135-46. doi: 10.1021/bi00375a020.
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.
已合成了四种新的溴乙酰胺基嘧啶核苷，它们是牛胰核糖核酸酶A（RNase A）活性位点的亲和标记物。所有这些核苷都能与该酶可逆结合并与之发生共价反应，导致酶失活。已测定了每种衍生物的结合常数Kb和一级分解速率常数k3。具体如下：3'-(溴乙酰胺基)-3'-脱氧尿苷，Kb = 0.062 M，k3 = 3.3×10⁻⁴ s⁻¹；2'-(溴乙酰胺基)-2'-脱氧木糖呋喃糖基尿嘧啶，Kb = 0.18 M，k3 = 1700×10⁻⁴ s⁻¹；3'-(溴乙酰胺基)-3'-脱氧阿拉伯糖呋喃糖基尿嘧啶，Kb = 0.038 M，k3 = 6.6×10⁻⁴ s⁻¹；以及3'-(溴乙酰胺基)-3'-脱氧胸苷，Kb = 0.094 M，k3 = 2.7×10⁻⁴ s⁻¹。3'-(溴乙酰胺基)-3'-脱氧尿苷仅与组氨酸-119残基反应，生成70%的在N-1位被取代的单烷基化产物、14%的在N-3位被取代的单烷基化衍生物以及16%的在N-1和N-3位均被取代的二烷基化产物。2'-(溴乙酰胺基)-2'-脱氧木糖呋喃糖基尿嘧啶和3'-(溴乙酰胺基)-3'-脱氧阿拉伯糖呋喃糖基尿嘧啶都绝对特异性地与组氨酸-12的N-3位反应。3'-(溴乙酰胺基)-3'-脱氧胸苷使组氨酸-12和-119烷基化。以57%的产率形成的主要产物在组氨酸-12的N-3位被取代。一种单烷基化衍生物，产率为8%，在组氨酸-119的N-1位被取代。一种二取代产物以14%的产率形成，在组氨酸-12的N-3位和组氨酸-119的N-1位均被烷基化。3'-(溴乙酰胺基)-3'-脱氧尿苷特有的“向下”2'-OH基团的特定相互作用，有助于使3'-溴乙酰胺基残基靠近组氨酸-119的咪唑环。3',5'-二核苷磷酸底物的2'-OH基团在催化机制中可能起类似作用，使组氨酸-119在转磷酸化步骤中使离去基团质子化。（溴乙酰胺基）核苷以多种不同构象结合在RNase A的活性位点，这导致了不同的特异性和烷基化速率。
