A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites
A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites
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DOI：
10.1016/s1074-5521(99)80125-2
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发表时间：
1999-11-01
影响因子：
--
通讯作者：
Sen, D
中科院分区：
文献类型：
--
作者：
Travascio, P;Bennet, AJ;Sen, D
背景资料：先前报道了使用体外选择方法衍生的18个核苷酸的DNA寡聚体PS2.M以亚微摩尔亲和力结合氯化血红素（Fe（III）-原卟啉IX）。DNA-氯化血红素复合物具有DNA增强的过氧化活性。PS2.M富含鸟嘌呤，需要钾离子折叠成其活性构象，与其形成鸟嘌呤四链体一致。在研究PS2.M的特异性催化特性时，我们测试了其RNA版本（rPS2.M）以及一个无关的DNA鸟嘌呤四链体（OXY 4）的过氧化特性。结果：rPS2.M的hemin结合亲和力比PS2.M弱30倍。然而，RNA-氯化血红素复合物的酶促过氧化的紫外-可见光谱和动力学与其DNA对应物几乎相同。两者都显示过氧化物酶活性大大大于血红素蛋白，如过氧化氢酶和铁（III）-肌红蛋白。动力学分析表明PS2.M和rPS2.M催化氯化血红素-过氧化氢共价复合物分解为产物。折叠的OXY 4的氯化血红素复合物（其与氯化血红素的结合强度与rPS 2.M一样强）具有明显的吸收光谱，并且仅具有高于背景水平的较小的过氧化物酶活性。结果进一步表明，只有一个子集的辅因子结合位点内形成的折叠核酸可能能够作为活性位点的功能，通过提供适当的化学环境催化。
Background: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2.M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-quaduplex. In investigating the specific catalytic features of PS2.M we tested the peroxidative properties of its RNA version (rPS2.M) as well as that of an unrelated DNA guanine-quadruplex, OXY4.Results: The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M. The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2.M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M) had a distinct absorption spectrum and only a minor peroxidase activity above the background level.The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.