Mundorff E C, Hanson M A, Varvak A, Ulrich H, Schultz P G, Stevens R C
Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA.
Biochemistry. 2000 Feb 1;39(4):627-32. doi: 10.1021/bi9924314.
Antibody AZ-28 was generated against the chairlike transition-state analogue (TSA) 1 and catalyzes the oxy-Cope rearrangement of substrate 2 to product 3. The germline precursor to AZ-28 catalyzes the reaction with a 35-fold higher rate (k(cat)/k(uncat) = 163 000), despite a 40-fold lower binding affinity for TSA.1 (K(D) = 670 nM). To determine the structural basis for the differences in the binding and catalytic properties of the germline and affinity-matured antibodies, the X-ray crystal structures of the unliganded and TSA.1 complex of antibody AZ-28 have been determined at 2.8 and 2.6 A resolution, respectively; the structures of the unliganded and TSA.1 complex of the germline precursor to AZ-28 were both determined at 2. 0 A resolution. In the affinity-matured antibody.hapten complex the TSA is fixed in a catalytically unfavorable conformation by a combination of van der Waals and hydrogen-bonding interactions. The 2- and 5-phenyl substituents of TSA.1 are almost perpendicular to the cyclohexyl ring, leading to decreased orbital overlap and decreased stabilization of the putative transition state. The active site of the germline antibody appears to have an increased degree of flexibility-CDRH3 moves 4.9 A outward from the active site upon binding of TSA.1. We suggest that this conformational flexibility in the germline antibody, which results in a lower binding affinity for TSA.1, allows dynamic changes in the dihedral angle of the 2-phenyl substituent along the reaction coordinate. These conformational changes in turn lead to enhanced orbital overlap and increased catalytic rate. These studies suggest that protein and substrate dynamics play a key role in this antibody-catalyzed reaction.
抗体AZ - 28是针对椅状过渡态类似物（TSA）1产生的，它催化底物2进行氧杂-Cope重排反应生成产物3。AZ - 28的种系前体催化该反应的速率要高35倍（k(cat)/k(uncat) = 163000），尽管其对TSA.1的结合亲和力低40倍（K(D) = 670 nM）。为了确定种系抗体和亲和力成熟抗体在结合及催化特性上存在差异的结构基础，分别以2.8 Å和2.6 Å的分辨率测定了未结合配体的抗体AZ - 28以及与TSA.1形成复合物的X射线晶体结构；AZ - 28种系前体未结合配体及与TSA.1形成复合物的结构均以2.0 Å的分辨率测定。在亲和力成熟的抗体-半抗原复合物中，TSA通过范德华力和氢键相互作用的组合被固定在催化不利的构象中。TSA.1的2-苯基和5-苯基取代基几乎与环己基环垂直，导致轨道重叠减少以及假定过渡态的稳定性降低。种系抗体的活性位点似乎具有更高程度的灵活性——结合TSA.1后，互补决定区H3（CDRH3）从活性位点向外移动4.9 Å。我们认为种系抗体中的这种构象灵活性导致其对TSA.1的结合亲和力较低，但却允许2-苯基取代基的二面角沿反应坐标发生动态变化。这些构象变化进而导致轨道重叠增强和催化速率提高。这些研究表明蛋白质和底物动力学在这种抗体催化反应中起关键作用。
