Kristensen O, Vassylyev D G, Tanaka F, Morikawa K, Fujii I
and Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, 565, Japan.
J Mol Biol. 1998 Aug 21;281(3):501-11. doi: 10.1006/jmbi.1998.1940.
The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 A. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency (kcat/kuncat=KS/KTSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (Nepsilon) of HisL27D is placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 A, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with TrpH100I and TyrH58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.
一种催化抗体6D9与过渡态类似物形成的复合物的三维结构已得到解析。该结构由两种不同的晶体形式确定，并在1.8埃的分辨率下进行了精修。用膦酸酯过渡态类似物3免疫诱导产生的抗体6D9，可将氯霉素单酯1的前药水解生成母体药物2。动力学研究表明，由于对过渡态的亲和力与催化效率之间的理论关系（kcat/kuncat = KS/KTSA），该抗体具有催化活性。晶体结构使这种理论关系可视化成为可能。HisL27D的一个侧链（Nε）处于关键位置，与过渡态类似物的膦酸酯氧形成氢键，距离为2.72埃，这表明在水解过渡态中与发展中的氧阴离子形成了氢键。在膦酸酯部分周围，除组氨酸外没有其他催化残基。此外，在抗体-半抗原复合物中，半抗原呈折叠构象，两个堆叠的芳香环通过与TrpH100I和TyrH58的芳香-芳香相互作用深埋在抗原结合位点中。结合半抗原的构象表明，抗体结合底物会将酯部分的构象改变为热力学不稳定的E型，从而使底物在催化过程中易于达到过渡态。这些观察结果表明，催化机制完全可以基于过渡态的稳定来解释。本文报道的精修高分辨率结构预计会对其他水解抗体的理解产生影响，因为它们的半抗原与本研究中使用的半抗原有一些独特的特征。
