| Enzyme is a highly efficient biocatalyst which can catalyze chemical reactions with substrate stereoselectivities and specificities under mild conditions. The design and synthesis of artificial catalysts with natural enzyme performance and the understanding of enzyme catalytic mechanism is one of the goals which a great many scientists are always pursuing. Glutathione peroxidases (GPx) are the well-known antioxidant selenoenzymes in organisms which can clear up several harmful hydroperoxides (ROOH) and then maintain the metabolic balance of reactive oxygen species (ROS) in vivo, thus protecting the biomembranes and other cellular components against oxidative damage. In certain pathogenic states, the production of ROS is enhanced and the excess ROS give a damage to various biomacromolecules such as RNA, DNA, protein, sugar and lipid, and therefore results in ROS-mediated diseases, including reperfusion injury, inflammatory process, age-related diseases neuronal apoptosis, cancer and cataract and so on. Therefore, GPx could be a candidate for antioxidant drugs. Unfortunately, scientists could not fully understand the structures of GPx as well as its catalytic mechanism in vivo at present. Fabrication of GPx models offers an ideal alternative for elucidating the origin of substrate binding interaction and catalytic mechanism of enzyme.By far, based on the knowledge of the structure of natural glutathione peroxidase and the understanding of the essence of enzyme catalysis (substrate binding and intermolecular catalysis), a great many of mimics have been successfully prepared by chemical and biological strategies, such as, 2,2′-Ditellurobis(2-deoxy-β-cyclodextrin), tellruo-subtilisin and telluro-GST and so on, all of which demonstrate excellent enzyme performance. However, during enzyme catalyzing, the binding for substrates and the forming enzyme-substrate comlplex and transition state is a dynamic process, then how to match the positions of the catalytic center and binding site in the enzyme model to facilitate the reaction proceed directionally, is our a new challenge for designing the enzyme model. Furthermore, for the GPx mimics with high catalytic activity, how to modulate the activity is another goal that we are pursuing.The flourishing development in nano and supramolecular science brings a new field in the design of artificial enzyme. Based on nano-scale materials, a great number of nanoenzyme models have been reported. Herein, based on the understanding of the structure of nature enzyme, we first choose micelle as a scaffold to construct a micellar enzyme model by supramolecular self-assembly. By studying its catalytic activity, the relationship between the catalytic center and binding site was well elucidated. Further, to better design an enzyme model in which the catalytic center and binding site were well matched, a surface imprinted polystyrene nanoparticle was constructed by molecular imprinting. As anticipated, the imprinted nanoenzyme model demonstrated high catalytic activity and substrate specificity. Finally, a temperature smart nanogel enzyme model was constructed by employing a temperature responsive N-isopropylacrylamide as a monomer. With the change of the temperature, the change of pore size and hydrophobicity play an important role in modulating the catalytic activity.1. Small molecular micelle enzyme modelMicelle has a self-assembly three-dimensional nanostructure and two distinct region─the hydrophobic interior core and hydrophilic charged surface. Its similarity to the structure of nature GPx catalytic center has attracted great attention. It was applied as a nanoreactor in hydrolysis and formation of double carbon-carbon bond has been widely reported. However, it have not been reported as GPx mimics yet. Herein, based on the structure of nature GPx, we synthesized a benzeneseleninic acid as a catalytic center and positive changed hexadecyltrimethylammonium bromide (CTAB) as a surfactant. In aqueous solution, they could self-assemble to form a micellar enzyme model. The catalytic activities were evaluated in both TNB and coupled reductase assayed system. It demonstrated high catalytic activity and substrate specificity. The experiments indicated that micelle is a good scaffold for constructing the GPx mimic and its hydrophobic interior core and positive charged surface played an important role for accelerating the enzyme-like reaction. Using this supramolecuar strategy to construct GPx mimic, considering the simple procedure and easy modulation, it is supposed to provide a new method to construct enzyme model.2. Polymeric micellar enzyme modelPrevious works have well demonstrated that the micelle enzyme model was a good GPx mimic. However, for small molecular micelle, normally it keeps a balance in aqueous solution. The weak stability dramatically limits its further application and the study of enzyme properties. So the double carbon carbon bond was introduced into the surfactant and catalytic center, and the micellar structure stability was enhanced by polymerizing. The obtained polymeric micellar enzyme model maintained the original micellar structure and demonstrated high catalytic activity and substrate specificity. In TNB assay system, using CUOOH as the other substrate, its catalytic activity is about 634000-fold enhancement compared with diphenlydiselenide. Furthermore, a serials of catalytic center monomers with various length were constructed. By polymerization, they were incorporated into various positions in the micelle. By comparising of their enzyme properties, we concluded that the match of the catalytic center and binding site played an important role in enhancing the catalytic activity. The enzyme model demonstrates the highest catalytic activity. when the catalytic center was designed on the rim of the micelle.3. Surface imprinted polystyrene nanoparticle enzyme modelTo make the catalytic center and binding site well match in a model and demonstrate high catalytic activity, molecular imprinting technique was employed and a surface imprinted polystyrene nanoparticle enzyme model was constructed. Based on the knowledge of natural GPx structure, a tellurium-containing compound and an arginine derivative were designed as catalytic center and binding site respectively in the model. As anticipated, this model demonstrated high catalytic activity. Through the study of the enzyme property, some conclusions are as follows: (1) for the design of the enzyme model, substrate binding is necessary, but the position match of the catalytic center and binding site is another important factor; (2) molecular imprinting is an effective technique for constructing enzyme model; (3) using intermediate of enzyme cycle as a template molecule reduces the imprinting procedure largely, and the surface imprinting overcomes the disadvantages of traditional imprinting, such as the transmogrification of the imprinted structure after removing the templates and the bad substrate permeation and so on.4. Nanogel enzyme model with temperature responsive catalytic activityTo realize the modulation of the catalytic activity and also the double control of the function of ROS, a temperature responsive nanogel enzyme model was constructed for the first time. The temperature responsive N-isopropylacrylamide (NIPAAm) was employed as a functional monomer, and a synthesized telluro-containing compound served both as catalytic center and cross-linker. By microemulsion polymerizing, the enzyme model was obtained. The three dimensional netlike structure of the nanogel interior core played a key role for the high catalytic activity. In TNS assay system, it demonstrated the highest catalytic activity at 32°C, and when the temperature was above 50°C, the nanogel nearly lost its catalytic activity. The experiment confirmed that the change of the pore size and the hydrophobicity of the nanogel interior core with the increased temperature played an important role in modulating the catalytic activity.
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