利用分子机器的组装与分解构建pH敏感性谷胱甘肽过氧化物人工酶

doi:10.11949/0438-1157.20220207

摘要/Abstract

摘要：

谷胱甘肽过氧化物酶（GPx）是体内一种重要的抗氧化酶，对GPx的人工模拟能够拓展其体外应用，利用生命体内天然pH差异实现响应的pH敏感性GPx人工酶更是有着广泛的潜在应用。然而目前制备的pH敏感性GPx人工酶普遍存在活性较低的缺点，本文设计合成了同时含有高活性GPx催化中心和两个伯胺基团的乙胺单碲醚分子，酶促反应动力学的分析结果表明其二级反应速率常数高达10¹ L·mol^-1·min^-1数量级。pH=6时，乙胺单碲醚分子能够与葫芦[6]脲分子（CB[6]）组装形成分子机器，此时活性中心被包埋于CB[6]的疏水空腔之中，仅能展现出（0.04±0.02）μmol·min^-1·μmol^-1的活性；pH=7时，分子机器部分分解，能够展现出高达（0.35±0.06）μmol·min^-1·μmol^-1的活性。因此，通过调控体系pH在6和7之间变化，借助分子机器的组装及部分分解调控人工酶活性的关闭与开启，从而构建了高活性的pH敏感性GPx人工酶。

关键词: 酶, 谷胱甘肽过氧化物酶, 催化, 生物催化, 分子机器, pH敏感性

Abstract:

Glutathione peroxidase (GPx) is an antioxidant enzyme in vivo. For expanding the application of GPx in vitro, imitating GPx functions has aroused great interest. The pH-sensitive GPx artificial enzymes that utilize natural pH differences in living organisms to achieve responses have a wide range of potential applications. However, up to now, the activities of pH sensitive GPx mimics are relatively low. Thus, organic tellurium compound I, which contains high activity catalytic center of glutathione peroxidase (GPx) and two primary amine groups, was designed and synthesized in this research. The enzymatic reaction kinetics measurement results demonstrated that the second order reaction rate constant of compound I was at the level of as high as 10¹ L·mol^-1·min^-1. Compound I formed molecular machine with CB[6] at pH=6. In this case, only (0.04±0.02) μmol·min^-1·μmol^-1GPx activity was shown as the active site was encapsulated into CB[6] and thereby cannot bind substrate. Molecular machine was partly dissociation when only one of the two primary amine groups was protonated at pH=7. In this case, (0.35±0.06)μmol·min^-1·μmol^-1GPx activity was shown as the active site gradually exposed to solution. The high activity pH sensitive artificial glutathione peroxidase was constructed through the formation and partly dissociation of molecular machine formed by compound I and CB[6] by changing pH between 6 and 7.

Key words: enzyme, glutathione peroxidase, catalysis, biocatalysis, molecular machine, pH sensitive

中图分类号:

Q 554⁺.6

安绍杰, 许洪峰, 李思, 许远航, 李佳锡. 利用分子机器的组装与分解构建pH敏感性谷胱甘肽过氧化物人工酶[J]. 化工学报, 2022, 73(8): 3669-3678.

Shaojie AN, Hongfeng XU, Si LI, Yuanhang XU, Jiaxi LI. Construction of pH sensitive artificial glutathione peroxidase based on the formation and dissociation of molecular machine[J]. CIESC Journal, 2022, 73(8): 3669-3678.

图/表 12

图1 利用分子机器的组装与分解构建pH敏感性谷胱甘肽过氧化物人工酶示意图

Fig.1 Schematic representation of pH sensitive artificial glutathione peroxidase based on the formation and dissociation of molecular machine

图2 乙胺单碲醚（化合物I）的合成路线图

Fig.2 Synthesis procedures of compound I

图3 化合物Ⅰ浓度分别为0、1、2、4 μmol·L-1条件下的催化曲线concentrations of compound Ⅰ/(μmol·L-1): a—0; b—1; c—2; d—4

Fig.3 Catalytic curves of different concentrations of compound Ⅰ by TNB assay system and GSH reductase-reduced NADPH coupled catalytic activity assay system at pH=7

图4 化合物Ⅰ在不同pH下的GPx活性

Fig.4 Catalytic activities of compound Ⅰ at different pHs

图5 化合物Ⅰ催化GSH还原H2O2的双倒数曲线

Fig.5 Double-reciprocal plots of the reduction of H2O2 by GSH under the catalysis of compound Ⅰ

图6 化合物Ⅰ可能的催化循环示意图

Fig.6 Proposed catalytic mechanism of compound Ⅰ

表1 化合物Ⅰ的表观动力学参数

Table 1 The apparent kinetic parameters of compound Ⅰ

[H₂O₂]/ (mmol·L^-1)	k_cat/min^-1	K_mGSH / (mol·L^-1)	(k_cat/K_mGSH)/ (L·mol^-1·min^-1)
0.33	7.96	2.65	3.00
0.5	6.68	2.48	2.69
0.75	6.51	2.63	2.48
[GSH]/ (mmol·L^-1)	k_cat/ min^-1	$K m H 2 O 2$ / (mol·L^-1)	(k_cat/ $K m H 2 O 2$ )/ (L·mol^-1·min^-1)
1.5	4.15	0.68	6.09
2	1.61	0.86	1.88
4	0.97	0.89	1.09

表1 化合物Ⅰ的表观动力学参数

Table 1 The apparent kinetic parameters of compound Ⅰ

[H₂O₂]/ (mmol·L^-1)	k_cat/min^-1	K_mGSH / (mol·L^-1)	(k_cat/K_mGSH)/ (L·mol^-1·min^-1)
0.33	7.96	2.65	3.00
0.5	6.68	2.48	2.69
0.75	6.51	2.63	2.48
[GSH]/ (mmol·L^-1)	k_cat/ min^-1	$K m H 2 O 2$ / (mol·L^-1)	(k_cat/ $K m H 2 O 2$ )/ (L·mol^-1·min^-1)
1.5	4.15	0.68	6.09
2	1.61	0.86	1.88
4	0.97	0.89	1.09

图7 化合物Ⅰ (5 mmol·L-1) 在pH=6时与不同浓度CB[6]混合物的部分1H NMR谱图concentration of CB[6] /(mmol·L-1): a—5; b—3.75; c—2.5; d—1.25

Fig.7 Partial 1H NMR spectra of mixtures of compound Ⅰ(5 mmol·L-1) with different amounts of CB[6] at pH=6

图8 化合物 Ⅰ (5 mmol·L-1)与 CB[6] (5 mmol·L-1) 在不同pH下的部分1 H NMR 谱图(500 MHz)

Fig.8 Partial 1H NMR spectra (500 MHz) of mixtures of compound Ⅰ (5 mmol·L-1) with CB[6] (5 mmol·L-1) at different pH

图9 pH=6时4 μmol·L-1化合物Ⅰ与空白（a）,及浓度分别为4 μmol·L-1（b）、3 μmol·L-1（c）、2 μmol·L-1（d）、1 μmol·L-1（e）、0 μmol·L-1（f）的 CB[6]混合物的催化曲线

Fig.9 Catalytic curves of mixture of 4 μmol·L-1 compound Ⅰ and blank (a), 4 μmol·L-1 (b), 3 μmol·L-1 (c), 2 μmol·L-1 (d), 1 μmol·L-1 (e), 0 μmol·L-1 (f) CB[6] at pH=6

图10 分子机器的催化曲线a—空白; b—4 μmol·L-1化合物Ⅰ与CB[6]1∶1混合物; c—4 μmol·L-1化合物Ⅰ

Fig.10 Catalytic curves of molecular machinea—blank; b—4 μmol·L-1 mixture of compound Ⅰ and CB[6] at the ratio of 1∶1; c—4 μmol·L-1 compound Ⅰ

图11 pH在6～9时分子机器催化活性的相对值(a)及分子机器催化活性的绝对值(b)

Fig.11 Relative catalytic activities (a) and catalytic activities (b) of the molecular machine at a range of pH from 6 to 9

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