CONSTRUCTION OF A NOVEL GUEST BIOMIMETIC GLUTATHIONE PEROXIDASE WITH SOLVENT-DEPENDENT CATALYTIC BEHAVIOR BY INCORPORATING THE ACTIVE CENTER INTO ADAMANTYL MOLECULE
© 2014 Yanzhen Yin",b, Chao Langc, Xiaoxi Hu", Zhongfeng Shi", Yun W&ng", Shufei Jiao", #, Chengxiang Cai", Junqiu Liuc
aSchool of Chemistry and Chemical Engineering, Qinzhou University, No. 89, Xihuan Nanlu, Qinzhou,
535000 P.R. China
bGuangxi Experiment Centre of Science and Technology, Guangxi University, Nanning, 530004 P.R. China cState Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, 130012 P.R. China Received May 22, 2013; in final form, August 1, 2013
A novel guest biomimetic glutathione peroxidase (3,3'-tellurobis(propane-3,1-diyl) diadamantanecarboxy-late, denoted as ADA-Te-ADA) was synthesized. ADA-Te-ADA functioned to overcome the disadvantages in the construction of building block for giant supramolecular biomimetic enzyme. To reveal the catalytic property of hydrophobic ADA-Te-ADA, the catalytic mechanism was investigated using PBS (phosphate buffer (pH 7.0. 50 mM))/methanol solvent mixture as assay solution. It indicated that ADA-Te-ADA exhibited typical enzyme catalytic behavior by saturation kinetics measurement. Importantly, ADA-Te-ADA exhibited the typical solvent-dependent catalytic behavior. And the highest catalytic rate 4.29 ^M x min-1 was obtained when the volume ratio of PBs: methanol was 5 : 5. Especially, the catalytic rates obtained based on various substrates proved that ADA-Te-ADA slightly displayed special substrate selectivity, which was the ideal catalytic characterization of building block for giant supramolecular biomimetic enzyme. The successfully synthesis of ADA-Te-ADA might highlight for the understanding of the catalytic mechanism of hydrophobic guest biomimetic glutathione peroxidase. And it also might provide the basement for the construction of giant supramolecular biomimetic enzyme.
Keywords: biomimetic enzymes, biocatalysis, biomimetics, enzyme activity, glutathione peroxidase.
DOI: 10.7868/S013234231401014X
1. INTRODUCTION
Overproduced reactive oxygen species (ROS) usually lead to many human oxidative stress-related diseases. Generally, the overproduced ROS are cleared by antioxidative defense system, especially by antioxidative enzyme system. Typically, glutathione peroxidase (GPx, EC 1.11.1.9) is an important selenium-containing enzyme among the antioxidative enzyme system [1—4]. Owing to its biologically crucial role, considerable strategies have been explored to produce biomimetic GPx with excellent catalytic ability [5—18]. In our group, based on the understanding of the structure of GPx, some biomimetic GPx have been de-
Abbreviations: GPx, glutathione peroxidase; ADA-Te-ADA, 3,3'-tellurobis(propane-3,1-diyl) diadamantanecarboxylate; ROS, reactive oxygen species; CUOOH, hydroperoxide; GSH, glutathione; NBT, 4-nitrothiophenol; TNB, 3-carboxyl-4-ni-trobenzenethiol. # Corresponding author (phone: 0086+07772807716; e-mail: jiaoshufei2013@163.com).
signed employing synthesis and supramolecular self-assembled strategy [9, 10, 19-25].
Recently, combining the self-assembled strategy and the concept of supramolecular chemistry, various assembled materials were well developed. Typically, supramolecular lipid ensembles of lipoporphyrins [26], self-assembling nanostructure of peptides [27], self-assembled supramolecular structures of oligogly-cylamides H-(Gly)m-NH2 (m = 3-5) [28], immobilized glutathione by complementary coordination binding [29], and biodegradable microcapsules by a self-healing of porous microspheres [30] were investigated by researchers. Especially, the constructions of biomimetic GPx based on synthesis and supramolecular strategy have attracted continuous focuses. Liu and coworkers pioneered in preparing 2,2'-diseleno-bis-P-cyclodextrin [31] and 2,2'-ditelluro-bis-P-cy-clodextrin [32] using organic synthesis method. The biomimetic synthesis of host cyclodextrin GPx based on the concept of supramolecular chemistry opened a new window for the construction of giant supramolec-
ular biomimetic GPx. Inspired by the host cyclodex-trin GPx, various self-assembled biomimetic GPxs were constructed based on variable supramolecular scaffold, such as vesicle [10, 23], nanotube [12], micelle [21], hyperbranched polymer [24, 25], and so on. It proved that such biomimetic GPxs exhibited the advantages of simplified synthesis [9, 33], controlled preparation [19] and variable catalytic cooperativity [22]. Significantly, the self-assembled biomimetic GPxs using host cyclodextrin as building block exhibited dramatically assembly and catalytic behavior [12, 24, 25, 33]. Not only was various assembly structure obtained, but also the remarkable catalytic rates, the stronger substrates binding ability and the better catalytic cooperativity were anchieved.
Nevertheless, two disadvantages have largely limited the further development of self-assembled biomimetic GPx. Firstly, one of the excellent propertis of host cyclodextrin GPx was buried as the hydrophobic cave of the cyclodextrin was usually used to complex the adamantyl guest molecule. Commonly, the hydrophobic cave of host cyclodextrin GPx could greatly enhance the catalytic rate and substrates binding ability. Secondly, the synthesis process of the host cyclodextrin GPx accompanied with the disadvantages of complicated synthetic route, high cost and low productivity. Therefore, to explore the simple and efficient biomimetic synthesis strategy of building block with GPx catalytic ability for self-assembled biomimetic GPxs still is a significant goal. The investigation might overcome the two disadvantages mentioned above and provide the significant alternative for host cyclodextrin GPx.
To meet such significant challenge, the exploration of the simple and efficient biomimetic synthesis of building block was carried out in this work. The GPx active site (tellurium) was successfully incorporated into a hydrophobic guest molecule (ADA-Te-ADA). This
study also boded well for the understanding of the catalytic mechanism of hydrophobic biomimetic GPx.
2. RESULTS AND DISCUSSION 2.1. The Design and the Preparation of ADA-Te-ADA
The crystal structure of bovine erythrocyte glu-tathione peroxidase was reported by Epp et al. in 1983 [34]. Up to now, various biomimetic GPxs were constructed using chemical strategy [5—7, 15], genetic engineering strategy [8, 13] and supramolecular strategy [12, 18, 21—25]. It illustrated that catalytic selenium center [10, 11, 13, 15], hydrophobic cavity [17, 18, 31, 32] and recognition site [21—25] were three important catalytic factors for efficient GPx catalytic activity. Particularly, the hydrophobic cave of host cyclodextrin GPx could greatly enhance the catalytic rate and substrates binding ability [17, 18, 31, 32]. However, the buried binding ability and the disadvantage in synthetic process existed in host cyclodextrin GPx have extremely limited the further development of self-assembled biomimetic GPx. Therefore, the significant goal in this work was the exploration of simple and efficient biomimetic synthesis strategy for building block with GPx catalytic ability. Usually, the traditional catalytic factors(recognition site and hydrophobic cavity) could be simply and efficiently anchored to biomimetic GPx by supramolecular self-assembled method [10, 12, 21—25]. Contrarily, the anchoring of such catalytic factors by traditional organic synthesis was complicated [4—7, 35]. Therefore, as an ideal building block for supramolecular assembled biomimetic GPx, it should provide the catalytic center and avoid traditional catalytic factors. Thus, based on the considerations mentioned above, the guest biomimetic GPx was designed and denoted as ADA-Te-ADA. And the synthetic route was depicted in Scheme 1.
Te + NaBH4 + H2O
Na2Te + H3BO3 + H2
Br'
"OH + Na2Te
HO
Te
OH
O
OH SOCl2
O
THF/NET3
Cl
HO
Te
OH
O
O
ADA-Te-ADA
Scheme 1. Synthetic route of ADA-Te-ADA.
In order to confirm the structure of ADA-Te-ADA, the H1 NMR measurement was carried in CDCl3. And
NMR spectrum was illustrated in Fig. 1. The peaks a, b, and c were deriving from the protons of adamantly
A
A
A
b
4.0 3.5 3.0 2.5 2.0
ppm
Fig. 1. NMR spectrum of ADA-Te-ADA in CDCl3 of 0.03 mM.
i e i ____A
t-1-1-1-1
group. And the detailed description of the corresponding relationship between peaks a, b, and c and the protons of adamantly group were noted using little arrows around the molecular structure of ADA-Te-ADA. Furthermore, the protons of — COOCH2—, —TeCH2—, and —CH2— were related to the peaks f, e, and d, respectively. It proved that ADA-Te-ADA was prepared successfully, which provided the basement for the fu-terther investigation of the cataytic mechanism of building block for giant supramolecular biomimetic enzyme.
2.2. The Characterization of the Catalytic Behavior ofADA-Te-ADA
As shown in Scheme 1, ADA-Te-ADA consisted of several hydrophobic groups, the solubility of ADA-Te-ADA in water was poor. It limited the traditionally determination of GPx catalytic ability in PBS. Considering that methanol was proved to be the appropriate co-solvent for hydrophobic dendrimer in previous re-
search [36], the solvent mixture consisting of PBS and methanol was adopted to determine GPx catalytic ability. As the solubility of biomimetic GPx and optical transmittances of its solution could affect the GPx catalytic activity [10, 21, 23], the optical transmittances determination of solvent mixture containing ADA-Te-ADA were carried out. It was employed to investigate the solubility of ADA-Te-ADA. The optical transmittances for the solvent mixture with the volume ratios of 9 : 1; 8 : 2; 7 : 3; 6 : 4; 5 : 5, 4 : 6 were 60.82, 83.504, 97.32, 97.503, 97.545, 97.536, respectively. And the typical histogram was illustrated in Fig. 2.
As shown in Fig. 2, the solvent mixture of ADA-Te-ADA trended to be a fixed op
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.