【作者】 薛超彬；

【导师】 罗万春；

【作者基本信息】 山东农业大学 ， 农药学， 2007， 博士

【摘要】 酚氧化酶(Phenoloxidase,PO)(EC.1.14.18.1)又称酪氨酸酶(Tyrosinase),是结构复杂的多亚基的含铜氧化还原酶。它广泛存在于动物、植物和微生物等生物体内,与人体的衰老、伤口愈合、果蔬的褐变等有密切关系。它是昆虫体内的一种重要酶类,在昆虫的变态发育和免疫系统中起着重要作用。由于酚氧化酶具有重要的理论意义和发展前景,大量的研究工作集中在此领域,筛选、设计、合成酚氧化酶抑制剂成为研究热点。在前期研究的基础上,本文以菜青虫(Pieris rapae L.)酚氧化酶为试验对象,测定了5,7,4’-三羟基黄酮等化合物对菜青虫幼虫生长发育的影响;选取铜铁试剂等化合物为酶抑制剂,研究它们对酚氧化酶的抑制作用;采用我们课题组自主设计、合成的3-羟基-4-甲氧基苯甲醛缩氨基硫脲等化合物为效应物,研究了它们对菜青虫酚氧化酶的抑制动力学,并建立了抑制作用模型;根据测得的抑制活性结果,进行了定量的结构与活性关系的研究,获得了具有较强预测能力的构效关系模型;应用FlexX法将抑制剂小分子与酚氧化酶活性位点成功进行了分子对接。本文的主要研究内容和结果如下。1.选用5,7,4’-三羟基黄酮、槲皮素、芦丁、5-甲氧基水杨酸和曲酸五种化合物,采用触杀和摄食毒力法进行了生物活性测定,研究生物源化合物对菜青虫幼虫生长发育的影响,结果表明,5,7,4’-三羟基黄酮和槲皮素对菜青虫幼虫生长发育具有明显的影响,浸渍法处理后72 h,两种化合物对试虫致死LC50值分别为0.226和0.951 g/L;叶片药膜法处理后72 h,LC50值分别为0.062和2.420 g/L。采用叶片药膜法时,当5,7,4’-三羟基黄酮的浓度高于0.200 g/L时,所有试虫均不能正常化蛹;采用浸渍法时,与对照相比,用5,7,4’-三羟基黄酮、槲皮素、芦丁、5-甲氧基水杨酸和曲酸处理的试虫5龄幼虫体重增长量明显降低。2.对菜青虫酚氧化酶进行初步分离纯化,并研究了基本酶学特性。研究结果表明:酶活力存在于35%饱和度硫酸铵的沉淀中,得率为69.52%,再经Sephadex G-100凝胶过滤层析进一步纯化,测得比活力为粗酶的6.22倍,得率为42.50%。研究部分纯化的菜青虫酚氧化酶的基本特性得出,该酶的最适pH值为7.0, pH在6.5～7.4范围内酶保持稳定的活力。最适温度为42℃,当温度低于32℃时,酶具有稳定的活力。研究金属离子对酶活力的影响,结果表明Na+和K+对酶活力没有影响;Cu2+在0～0.100 mmol/L范围内对酶活力表现激活作用,浓度大于0.125 mmol/L时表现抑制作用,其IC50为0.651 mmol/L。3.测定了4-己基间苯二酚、4-十二烷基间苯二酚、铜铁试剂、5,7,4’-三羟基黄酮和槲皮素对酶活力的抑制作用,并探讨了其抑制机理。研究结果表明:4-己基间苯二酚和4-十二烷基间苯二酚对酶表现可逆抑制效应,均为竞争型抑制类型,其IC50分别为1.50μmol/L和1.12μmol/L,抑制常数KI分别为0.50μmol/L和0.47μmol/L;铜铁试剂对酶表现为可逆抑制效应,为竞争型抑制类型,其IC50为0.10 mmol/L,KI为0.076 mmol/L;5,7,4’-三羟基黄酮和槲皮素对菜青虫酚氧化酶具有明显的抑制作用, IC50分别为25.65 mg/L和43.94 mg/L;研究不同浓度铜离子对该酶的影响,结果表明Cu2+在0～0.100 mmol/L范围内对酶活力表现激活作用,浓度大于0.125 mmol/L时表现抑制作用,其IC50为0.651 mmol/L。4.以L-酪氨酸和L-DOPA为底物,在氧饱和的条件下测定酚氧化酶氧化底物的动力学过程;测定抑制剂3-羟基-4-甲氧基苯甲醛缩氨基硫脲(3-H-4-MBT)和2-羟基苯甲醛(2-HBD)对酚氧化酶单酚酶和二酚酶活性的影响,研究结果表明:3-H-4-MBT和2-HBD均能显著抑制单酚酶和二酚酶活性,在酶的测活体系中加入3-H-4-MBT或2-HBD,二者均能显著延长反应体系到达稳态的时间,即这两种化合物均可延长酶反应的迟滞时间。研究结果显示3-H-4-MBT和2-HBD均为可逆的非竞争性抑制剂,3-H-4-MBT对单酚酶和二酚酶的IC50分别为0.14±0.02μmol/L和0.26±0.04μmol/L,对二酚酶的抑制常数KI (KI=KIS)为0.30μmol/L;2-HBD对单酚酶和二酚酶的IC50分别为8.08±0.11 mmol/L和4.14±0.08 mmol/L,对二酚酶的抑制常数KI (KI=KIS)为1.21 mmol/L。在不同浓度的3-H-4-MBT(或2-HBD)测活体系中,测定酚氧化酶氧化底物L-DOPA的动力学过程,随着反应时间的增加,反应体系逐渐到达稳态,此时反应历程为一直线。根据邹氏动力学原理,建立了3-H-4-MBT(2-HBD)对酚氧化酶的抑制模型,并求得抑制反应速率常数k-0和k+0。5.定量构效关系(QSAR)是农药分子设计中很重要的方法,本文详细介绍了2D-QSAR中的Hansch-Fujita法及3D-QSAR中的比较分子力场分析(CoMFA)法和比较分子相似指数分析(CoMSIA)法,并运用上述三种方法对酚氧化酶抑制剂进行了构效关系研究。运用Hansch-Fujita法对苯甲醛类、苯甲酸类和苯甲醛缩氨基硫脲类三组化合物进行了2D-QSAR研究,其中采用了Hammett电子效应参数σ、疏水性参数clogP、立体效应参数MR和氢键受体共四个参数,研究结果表明,苯甲酸类和苯甲醛缩氨基硫脲类两组化合物的构效关系特点比较相似,这也说明此两类化合物很可能作用在酶的同一位点,并且氢键受体和立体效应参数是影响化合物抑制活性的两大因子;而苯甲醛类化合物的构效关系不同于上述两组化合物,影响该组化合物生物活性的因子主要是疏水性参数clogP。应用CoMFA和CoMSIA两种方法,采用公共亚结构基础的叠合原则,进行了三维定量构效关系(3D-QSAR)研究,研究结果表明,构建的CoMFA和CoMSIA模型具有很好的预测能力,统计结果可靠。CoMFA模型中使用了6个主成分,模型的交叉验证系数q2=0.926,预测标准误(SEE)为0.250,非交叉验证系数r2=0.986。CoMSIA模型的建立采用了立体场(steric)、静电场(electrostatic)、疏水场(hydrophobic)、氢键受体场(hydrogen bond acceptor)和氢键供体场(hydrogen bond donor)五种场的自由组合。当模型采用立体场、疏水场和氢键受体场三个场时,模型的交叉验证系数q2=0.933,非交叉验证系数r2=0.984,F值和SEE值分别为381.764和0.271。因此,获得的3D-QSAR模型将为设计新颖、高活性的酚氧化酶抑制剂提供理论指导。6.本文采用FlexX法,以抑制剂小分子为配体,以抗生链酶菌(S. castaneoglobisporus)酚氧化酶晶体(PDB:1WX2)为受体进行了分子对接。研究结果表明:用FlexX法将抑制剂小分子成功对接到酶的活性中心,抑制剂在酶活性位点的结合方式有三种,第一种结合方式是苯甲醛缩氨基硫脲类化合物与酶活性位点的结合,其主要结合模式为化合物上缩氨基硫脲结构中的“夹钳”(H21-N9-C10- N12-H22)结构与酪氨酸残基(Tyr98)上的氧原子形成氢键;第二种结合方式是苯甲醛类化合物与酚氧化酶活性位点的结合,与苯甲醛缩氨基硫脲类化合物类似,该类化合物苯环结构的邻、间、对位上的羟基、以及相邻的氢原子形成一个“夹钳”结构,分别与Tyr98上的氧原子形成氢键;第三种结合方式是苯甲酸类化合物与酚氧化酶活性位点的结合,其结合方式为苯甲酸类化合物的羧基中的羟基与Tyr98上的氧原子形成氢键,羧基对位上的基团与氨基酸Trp184,Arg185和Pro102等发生氢键作用。更多还原

【Abstract】 Phenoloxidase (PO) (EC.1.14.18.1) also known as tyrosinase, is a structure-complex and multifunctional copper-containing enzyme, which widely distributed among animals, plants, fungi, and prokaryotes. PO is thought to be involved in many biological processes of invertebrates, such as consenescence of human body, wound healing, fruits and vegetables browning and pigment formation. It is one of the key enzymes in the development process of insects, the enzyme possesses an important function in metamorphism developing and immunity system. Currently, many studies focused on this field in order to screen, design and synthesis PO inhibitors for the importance theory of PO inhibitors and its bright future. In the present paper, compounds of 5, 7, 4’-trihydroxyflavone etc. were selected to investigate the effect of natural occurring compounds on the growth of Pieris rapae larvae, compounds cupferron etc. were selected as the PO inhibitors to determine the inhibitory effects against the enzyme, besides, compounds 3-hydroxy-4-methoxy benzaldehyde thiosemicarbazone (3-H-4-MBT) etc. were designed and synthesized in our laboratory, and the kinetics inhibition of PO were studied using these compounds.Additionally, the bioactivity results were used to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models using two molecular field analysis techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), robust and predictive 3D-QSAR models were obtained from CoMFA and CoMSIA. Furthermore, the molecular interactions between the ligands and the target were studied using a flexible docking method (FlexX) and the best scored candidates were docked flexibly.The contents and results were summarized as follows: 1. Compounds of 5, 7, 4’-trihydroxyflavone, quercetin, rutin, 5-methoxysalicylic acid and kojic acid were selected, in the present paper, to investigate the effect of natural occurring compounds on the growth of Pieris rapae larvae. Both dipping methods and ingestion methods were used in the bioassay, and the results showed that the LC50 value of the chemicals against the tested larva were estimated to be 0.226 and 0.951 g/L at 72 hours after treatment with dipping methods for 5, 7, 4’-trihydroxyflavone and quercetin, respectively, or 0.062 and 2.420 gl/L with ingestion methods, respectively. The results also indicted that all of the tested larva couldn’t pupated when the concentration of 5, 7, 4’-trihydroxyflavone beyond 0.200 g/L with ingestion methods, and, the increased amount of body weight of the 5th instar Pieris rapae larva, which were dipped in the solution of 5, 7, 4’-trihydroxyflavone, quercetin, rutin, 5-methoxysalicylic acid or kojic acid, were decreased obviously compared with the control.2. The results of PO purification showed that much of the enzyme activity was in the deposition of 35% saturated (NH4)2SO4, and the enzyme was purified 3.08-fold with a recovery of 69.52%. And then, PO was purified 6.22-fold with a recovery of 42.50% when the enzyme was chromatographed on Sephadex G-100 gel filtration. The properties of PO were determined, in the present paper, the results indicated that the optimum pH was 7.0 and the enzyme with a stable activity when the pH reaction system between 6.5~7.4. The optimum temperature was 42℃, and the enzyme with a stable activity when the temperature reaction system less than 32℃. Effects of some metal ions on the PO activity were studied, the results showed that Na+ and K+ had no effects on the enzyme activity. Meanwhile, the results showed that the PO activity was enhanced by Cu2+ when the concentration at 0～0.100 mmol/L, but the activity was inhibited by the same ion when the concentration went over to 0.125 mmol/L, and the IC50 was estimated to be 0.651 mmol/L.3. The inhibitory effects on the PO activity by 4-hexylresorcino, 4-n-dodecylresorcino, cupferron, 5, 7, 4’-trihydroxyflavone and quercetin were determined, and the possible mechanism of these inhibitors were discussed also. The results showed that 4-hexylresorcino and 4-n-dodecylresorcino were reversible competitive inhibitors on PO, and the IC50 were 1.50μmol/L and 1.12μmol/L, respectively, the inhibitory constants (KI) were also determined to be 0.50μmol/L and 0.47μmol/L, respectively. Cupferron was a reversible competitive inhibitor on the enzyme, the IC50 and the inhibitory constant (KI) was 0.10 mmol/L and 0.076 mmol/L, respectively. The results also indicated that 5, 7, 4’-trihydroxyflavone and quercetin could also inhibit the PO activity, and the IC50 were estimated to be 25.65 and 43.94 mg/L, respectively.4. In the present study, the kinetic assay in air-saturated solutions and the kinetic behavior of PO from P. rapae larvae in the oxidation of L-tyrosine (a monophenol) and L-DOPA (L-3, 4-dihydroxyphenylalanine) (a diphenol) was studied. The inhibitory effects of 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone (3-H-4-MBT) and 2-hydroxybenzaldehyde (2-HBD) on the monophenolase and diphenolase activities of PO were also studied in the present paper. The results showed that 3-H-4-MBT and 2-HBD can inhibit both the monophenolase and diphenolase activities of PO. The lag period of L-tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activities of the enzyme sharply decreased in the reaction course. Inhibitor 3-H-4-MBT was found to be noncompetitively reversible compound with a KI (KI=KIS) of 0.30μmol/L and an estimated IC50 of 0.14±0.02μmol/L for monophenolase or 0.26±0.04μmol/L for diphenolase. Inhibitor 2-HBD was found to be noncompetitively reversible with a KI (KI=KIS) of 1.21 mmol/L and an estimated IC50 of 8.08±0.11μmol/L for monophenolase or 4.14±0.08μmol/L for diphenolase. In the time course of the oxidation of L-DOPA catalyzed by the enzyme in the presence of different concentrations of 3-H-4-MBT or 2-HBD, the rate decreased with increasing time until a straight line was approached. The microscopic rate constants k-0 and k+0 for the reaction of 3-H-4-MBT or 2-HBD with the enzyme were determined.5. Quantitative structure-activity relationship (QSAR) studies are important approaches in the design of pesticidal molecules. The method of Hansch-Fujita of 2D-QSAR, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were introduced in detail in this paper, and the QSAR of PO inhibitors were carried out based on three methods above.In the present paper, the quantitative relationship between the structure of benzaldehyde derivatives, benzoic acid derivatives, benzaldehyde thiosemicarbazone derivatives and their inhibitory activities against P. rapae larvae PO were analyzed using Hansch-Fujita approach. The chemical descriptors, such as electronic parameters Hammettσ, hydrophobic parameters clogP, steric parameters MR and hydrogen bond acceptor were employed in this study. The results showed that the structure-activity relationships (SAR) of benzoic acid derivatives and benzaldehyde thiosemicarbazone derivatives were identical, and they may act on the same target site of the PO receptor. The hydrogen bond acceptor and steric parameters descriptors were the most significant factors on determining inhibitory activity of the two sets of compounds. The structure-activity relationships of benzaldehyde derivatives and benzaldehyde thiosemicarbazone derivatives indicated that the action mode on the PO receptor were different, or the action site of the two sets of compounds were different.The bioactivity results were used to construct 3D-QSAR models using two molecular field analysis techniques: CoMFA and CoMSIA. After carrying out superimposition using common substructure-based alignment, robust and predictive 3D-QSAR models were obtained from CoMFA (q2 = 0.926, r2 = 0.986, SEE = 0.250) and CoMSIA (q2 = 0.933, r2 = 0.984, F = 381.764, SEE = 0.271) with 6 optimum components. And, the steric field, hydrophobic field and hydrogen bond acceptor field were applied in the CoMSIA modle. The 3D-QSAR model built here will provide hints for the designing with novel phenoloxidase inhibitors.6. In the present study, the inhibitors were used as the ligands and the PO crystal structure (PDB: 1WX2) of S. castaneoglobisporus was employed as the target, and, the molecular interactions between the ligands and the target were studied using a flexible docking method (FlexX). The results showed that the best scored candidates were docked flexibly, and there are three interaction modes among the three compounds.The first interaction mode is the benzaldehyde thiosemicarbazone analogues interacted with the PO active site. The benzaldehyde thiosemicarbazone analogues contains a chain of atoms (H21 - N9 - C10 - N12 - H22) spatially arranged in what might be termed a“clamp”structure. The distance between both hydrogen atoms of the clamp and the carbonyl oxygen atom of Tyr98 is 1.991 ?, indicating the likely presence of a pair of hydrogen bonds. Formation of these two hydrogen bonds stabilizes the position of Tyr98, preventing Tyr98 from participating in the interaction between PO and ORF378. The second interaction mode is benzaldehyde analogues interacted with the PO active site, which is more like the first interaction mode, the hydrogen bonds were formed also. The third interaction mode is the benzoic acid derivatives interacted with the active site. Some hydrogen bonds were formed between the hydroxyl of carboxyl and oxygen of Tyr98, between the contraposition group of carboxyl and amino acid Trp184, Arg185 and Pro102.更多还原