【作者】 赵晓明；

【导师】 王丽萍；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 过氧化物酶对多种由于自由基损伤而引的疾病的预防和治疗有着重要意义,但由于天然过氧化物酶作为抗氧化药物的种种缺点,用模拟酶的办法来制备具有药用价值的过氧化物酶的模拟物成为了新的研究方向。本论文以过氧化物酶的辅基亚血红素为筛选靶目标,通过噬菌体表面展示肽库筛选出一系列的与亚血红素特异性结合的短肽配基,并得到了保守序列。以得到的序列为基础,设计合成了一系列的含有His和Arg的亚血红素—七肽化合物模拟酶,活性达到了天然微过氧化物酶MP-11活力的90%以上,其中DhHP-2RⅠ( Dh-AlaHisArgAlaAlaArg)的催化效率最高。对缺少His但包含Arg残基的序列进行了二级结构预测,根据预测结果合成了亚血红素—十二肽化合物模拟酶,其活性达到了天然微过氧化物酶MP-11活力的50%以上。以上的工作使我们进一步确定了Arg是参与过氧化物酶活性中心的重要氨基酸,为今后设计活性更高的过氧化物模拟酶打下了基础。更多还原

【Abstract】 Peroxidases, which exist in the animals, plants and aerobic microorganism,cooperate with other antioxidants to balance the metabolization of free radicals. Innormal physiological situation, nearly all of the aerobic livings can produce freeradicals in the process of utilizing oxygen. It is essential for free radicals to supportthe living activities in low concentration of physiological level. In abnormalphysiological situation, bodies either produce more free radicals or can’t get rid ofadequate free radicals, which break up the normal balance, then result in thedamage of cells. In spite of the significance of the peroxidases in the prevention and therapy ofthe diseases caused by free radicals, the therapeutical potential of naturalperoxidases is severely limited by its instability, cost, and other factors;thusenzyme mimetics of peroxidases are desirable. There are three kinds of enzymemimetics: simple organic compounds, peptide mimetics, and protein mimetics.Compared with simple organic compounds, peptide mimetics have the advantagesof low toxicity, high activity and easy synthesis. At the same time, compared withprotein mimetics, peptide mimetics have the low molecular weight and highstability. Also the modern technology makes it possible to synthesize peptide ofany sequences.Ascorbate peroxidase is a heme-protein that plays a central role in scavengingH2O2-dependent oxidation of both ascorbate and various aromatic substrates invivo. Fe3+ of hemin is the prerequisite for the enzyme activity of natural ascorbateperoxidase. Peptides MP-8, MP-9 and MP-11, the enzyme cleavage products ofcytochrome C (CytC), have been found to be good peroxidase mimetics;theiranti-oxidation has been proven in an induced cataract model;and they have beenextensively studied as peroxidase mimetics. These peptides contain one histidine,and they are covalently bonded with hemin. This specific covalent binding,however, makes it difficult to chemically synthesize this kind of peptide mimetics,very likely due to the poor affinity between the peptides and hemin. The resultedand hemin-coupled peptide mimetics are generally unstable;they tend to have abad solubility;and thus their applications are quite limited.In this thesis, the deuterohemin take the place of the hemin of the naturalascorate peroxidase mainly because the alkenyls in the native enzyme can beeasily oxidized by peroxide, the substrate. The phage display peptide library wasused to screen the small peptide ligand of deuteronhemin, and then the peptidemimic of peroxidases, which use the hemin as the prosthetic group, was carried out.After four rounds of screening, individual clones were isolated and sequenced, anda series of small peptide ligands with specific binding affinity to deuteroheminwere obtained. Among the screened peptide sequences, the peptides including Hisor Arg have the highest occurrence frequency. His is the prerequisite for theenzyme activity of natural ascorbate peroxidase. The carbimidine group in the sidechain of Arg contains five hydrogen donors, which usually participate in thecatalytic process of the enzyme. Among the screened peptide sequences, thepeptides that the length between deuterohemin and His is one residue have thehighest occurrence frequency. Our group had found that the optimum lengthbetween deuterohemin and His is one residue. Based upon this peptide sequence,two mimic of peroxidase was synthesized, and a relative high enzyme activity wasmeasured. In order to seek peptide peroxidase mimetics of better activity, we havesynthesized a series of DhHP-7 analogues and then the active sites of theperoxidase activity of DhHP-7 were elucidated: on the basis of the length betweendeuterohemin and His is one residue, this peroxidase mimic containing arginineresidue shows high peroxidase activity and Dh-AHARAAR shows the highest kcat/Km value.Previous studies have shown that the Mb mutants lacks the distal histidine butcontains an arginine residue that appears to be capable of stabilizing the binding ofligands to the iron center through hydrogen-bonding interactions from theguanidinium group. Among the screened peptide sequences, the peptides lacks thedistal histidine but contains an arginine residue have the higher occurrencefrequency. Secondary Structure Prediction have been done,and then two peptideswere chosen to research function of Arg. Studies show the peptides lacks the distalhistidine but contains an arginine residue have the higher enzyme activity.In this thesis, the phage display peptide library was used to screen the smallpeptide ligand of deuterohemin, and then the peptide mimic of peroxidases, whichuse the hemin as the prosthetic group, was carried out. The result listed abovemight be useful to study peroxidase mimetics that show the higher peroxidaseactivity.更多还原