Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor
Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor
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DOI：
10.1002/chem.200501613
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发表时间：
2006-09-25
影响因子：
4.3
通讯作者：
Niemeyer, Christof M.
中科院分区：
文献类型：
--
作者：
Fruk, Ljiljana;Mueller, Joachim;Niemeyer, Christof M.
重组的载脂蛋白酶与DNA修饰的血红素（原卟啉IX）辅因子已被用作一种工具，以产生人工酶，可以特异性地固定在固体表面。为此，共价血红素-DNA加合物的合成，并随后用于重组的载脂蛋白肌红蛋白（aMb）和载脂蛋白辣根过氧化物酶（aHRP）。重组产生催化活性酶，其中含有一个或两个DNA寡聚体偶联到酶的活性位点附近。这些DNA-酶结合物的动力学研究，进行了两个基板，ABTS和Amplex红，表现出显着增加的DNA-Mb酶的过氧化物酶活性，而减少酶活性观察到的DNA-HRP酶。所有DNA-酶缀合物都能够特异性结合到含有互补DNA寡聚体作为捕获探针的固体支持物上。通过DNA定向固定化方法固定化的酶的动力学分析显示，酶在与捕获寡聚物杂交后保持活性。通过DNA杂交实现的可编程结合性质使得此类半合成酶缀合物可用于广泛的应用，特别是在生物催化、电化学传感和作为生物材料的构建块中。
The reconstitution of apo enzymes with DNA oligonucleotide-modified heme (protoporphyrin IX) cofactors has been employed as a tool to produce artificial enzymes that can be specifically immobilized at the solid surfaces. To this end, covalent heme-DNA adducts were synthesized and subsequently used in the reconstitution of apo myoglobin (aMb) and apo horseradish peroxidase (aHRP). The reconstitution produced catalytically active enzymes that contained one or two DNA oligomers coupled to the enzyme in the close proximity to the active site. Kinetic studies of these DNA-enzyme conjugates, carried out with two substrates, ABTS and Amplex Red, showed a remarkable increase in peroxidase activity of the DNA-Mb enzymes while a decrease in enzymatic activity was observed for the DNA-HRP enzymes. All DNA-enzyme conjugates were capable of specific binding to a solid support containing complementary DNA oligomers as capture probes. Kinetic analysis of the enzymes immobilized by the DNA-directed immobilization method revealed that the enzymes remained active after hybridization to the capture oligomers. The programmable binding properties enabled by DNA hybridization make such semisynthetic enzyme conjugates useful for a broad range of applications, particularly in biocatalysis, electrochemical sensing, and as building blocks for biomaterials.