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

在目前的研究中，我们为两种催化剂、6D 9和9 C10进行了结构研究，将非生物活性氯苯酚单雌激素衍生物（基质）的羟基催化剂转化为生成氯苯酚。Although there is a high similarity in primary structures of these antibodies， 6D9 possesses the higher activity （kイD2catイD2/kイD2uncatイD2 = 895） than 9C10 does （kイD2catイD2/kイD2uncatイD2 = 56）。论酶研究的基础，论抗体在过渡状态模拟（TSA）的绑定常数中的差异，以及底物对于催化活性的表达是负责任的。因此，我们决定如何利用核磁共振在原子水平上利用核磁共振检测催化抗体。根据我们的核磁共振分析结果，我们将以下内容纳入其中：1。6D 9形式的His 27 d（L）的Imidazole质子将氢键与TSA磷酸盐组的氧原子结合起来，而在6D 9-Substrate，9 CIO-Sub中找不到氢键 ... More strate and 9 cIO-TSA复合体。His 27 d（L）的6D 9扮演了一个重要的角色来解释TSA和Substrate.2中化学结构之间的差异。在边界状态下，TSA和Substrate的芳香质子共振的不寻常的传播揭示了4-（三氟乙酰）氨基）苯基环的翻转运动在9 C10-Substrate和9 C10-TSA复合体中受到限制。同一现象在6D 9-TSA复合体中的TSA两个芳香环中被注意到，并指示在6D 9-TSA复合体中的氨基酸居民和两个芳香环之间存在强烈的相互作用。考虑到两个芳香环在绑定站点之间的相对位置是不同的，因为TSA和Substrate since the atomic Orbitals of carbonyl carbon in Substrate and phosphorus in TSA are spy D12型D1和spy D13型D1， Respectively，我们将考虑6D 9可以识别TSA和Substrate之间的结构差异，通过互动，然后6D 9获得高催化度下一步，我们进行了一项关于催化抗体7 C8的结构研究。在这项研究中，我们研究了反应中心的微环境，基质中的碳基碳与7 C8绑定，并确定了7 C8催化反应率的pH曲线。These results indicate that the residue of which PKa is approximately 4. 5 affManagement the catalytic mManagement ism of 7 C8。用7 C8标记的7 C8的pH滴定研究被揭示的功能安置为His 92（L）。它与His 92（L）和Tyr 95（H）在催化反应中发挥重要作用。对催化机制的调查是这些驻地现在正在进行中。Less（低）

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