NMR ANALYSIS OF STRUCTURAL CHARACTERISTICS OF CATALYTIC ANTIBODIES AND IMPROVEMENT OF THEIR CATALYTIC ACTIVITIES

催化抗体结构特征的NMR分析及其催化活性的提高

• 批准号: 10672018
• 负责人: TAKAHASHI Hideo
• 金额: $ 2.05万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672018/
• 关键词: CATALYTIC ANTIBODY; NMR; TRANSITION STATE ANALOG; SUBSTRATE; HYDROGEN BOND; Fv; 水素結合; ヒスチジン; 互変異性

In the present study we performed a structural study for two catalytic antibodies, 6D9 and 9C10, which catalyze the hydrolysis of a non-bioactive chloramphenicol monoester derivative (Substrate) to generate chloramphenicol. Although there is a high similarity in primary structures of these antibodies, 6D9 possesses the higher activity (kィイD2catィエD2/kィイD2uncatィエD2 = 895) than 9C10 does (kィイD2catィエD2/kィイD2uncatィエD2 = 56). On the basis of the enzymatic study, the difference in the binding constant of the antibodies for transition state analogue (TSA) and Substrate is responsible for the expression of the catalytic activities. Therefore we decided to investigate how the catalytic antibodies discriminate TSA from Substrate at the atomic level using NMR. From the result of our NMR analyses, we conclude the followings,1. Imidazole proton of His27d(L) of 6D9 forms a hydrogen bond to the oxygen atom of phosphonate group of TSA, while the hydrogen bond is not found in the 6D9-Substrate, 9CIO-Sub … More strate and 9CIO-TSA complexes. His27d(L) of 6D9 plays an important role for discriminating the difference of chemical structure between TSA and Substrate.2. Unusual broadening observed for aromatic proton resonances of TSA and Substrate in the bound state revealed that a flip-flop motion of 4-((trifluoroacetyl)amino)phenyl ring is restricted in the 9C10-Substrate and 9C10-TSA complexes. The same phenomenon is noted in both of the two aromatic rings of TSA in the 6D9-TSA complex, indicating that a strong interaction exists between the amino acid residues and both of the two aromatic rings in 6D9-TSA complex. Considering that relative positions of the two aromatic rings in the binding site is different between TSA and Substrate since the atomic orbitals of carbonyl carbon in Substrate and phosphorus in TSA are spィイD12ィエD1 and spィイD13ィエD1, respectively, we conclude that 6D9 could recognize the structural difference between TSA and Substrate in the bound state through the interaction and then 6D9 gain the high catalytic activities.Next, we performed a structural study of catalytic antibody 7C8. In this study we have investigated the microenvironment of the reaction center, carbonyl carbon of Substrate which is bound to 7C8, and determined the pH profile of the 7C8 catalyzed reaction rate. These results indicate that the residue of which pKa is approximately 4.5 affects the catalytic mechanism of 7C8. The pH titration study of 7C8 labeled with ィイD115ィエD1N revealed that the functional residue is His92(L). It is concluded that His92(L) as well as Tyr95(H) play a significant role in catalytic reaction by 7C8. Investigation of catalytic mechanism by these residues is now in progress. Less

在本研究中，我们对两种催化抗体 6D9 和 9C10 进行了结构研究，它们催化非生物活性氯霉素单酯衍生物（底物）水解生成氯霉素。尽管这些抗体的一级结构具有很高的相似性，但6D9的活性（kィイD2catィエD2/kィイD2uncatィエD2 = 895）比9C10（kィイD2catィエD2/kィイD2uncatィエD2 = 56）更高。根据酶学研究，过渡态类似物（TSA）和底物的抗体结合常数的差异是催化活性表达的原因。因此，我们决定研究催化抗体如何使用 NMR 在原子水平上区分 TSA 和底物。根据我们的核磁共振分析结果，我们得出以下结论：1。 6D9的His27d(L)的咪唑质子与TSA的膦酸基团的氧原子形成氢键，而6D9-Substrate、9CIO-Sub ... 更多strate和9CIO-TSA复合物中未发现氢键。 6D9的His27d(L)对于区分TSA和底物化学结构的差异具有重要作用。2．在结合状态下观察到 TSA 和底物的芳香质子共振异常展宽表明 4-((三氟乙酰基)氨基)苯环的翻转运动在 9C10-底物和 9C10-TSA 复合物中受到限制。在6D9-TSA复合物中TSA的两个芳香环中也观察到相同的现象，表明氨基酸残基与6D9-TSA复合物中的两个芳香环之间存在强相互作用。考虑到TSA和Substrate的结合位点中两个芳香环的相对位置不同，因为Substrate中的羰基碳和TSA中的磷的原子轨道分别为spィイD12ィエD1和spィイD13ィエD1，我们得出结论，6D9可以通过相互作用识别TSA和Substrate在结合状态下的结构差异，然后6D9获得 高催化活性。接下来，我们对催化抗体7C8进行了结构研究。在本研究中，我们研究了反应中心的微环境、与7C8结合的底物的羰基碳，并确定了7C8催化反应速率的pH曲线。这些结果表明pKa约为4.5的残基影响7C8的催化机制。对ィイD115ィエD1N标记的7C8进行pH滴定研究，结果表明功能残基为His92(L)。结论是His92(L)和Tyr95(H)在7C8的催化反应中起重要作用。这些残留物的催化机制的研究正在进行中。较少的

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