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首页> 外文期刊>Biochemistry >Conformational effects in biological catalysis: an antibody-catalyzed oxy-cope rearrangement.
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Conformational effects in biological catalysis: an antibody-catalyzed oxy-cope rearrangement.

机译：生物催化的构象效应：抗体催化的氧气 - 抗凝固重排。

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摘要

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.
机译：抗体AZ-28抵抗椅子升降状态模拟（TSA）1，并催化基材2的氧气凝固到产物3. AZ-28的种系前体催化与35倍较高速率的反应（K. （猫）/ k（Uncat）= 163 000），尽管对TSA.1（K（D）= 670nm）的较低结合亲和力等40倍。为了确定种系和亲和力成熟抗体的结合和催化性质差异的结构基础，抗体AZ-28的抗体和TSA.1复合物的X射线晶体结构已在2.8和2.6 a下测定分别分辨率;将芽底前体的已解粘和TSA.1复合物的结构均以2分辨率确定为2.0。在亲和力成熟的抗体中。通过范德华和氢键相互作用，HAPTEN复合物TSA通过组合在催化不利的构象中固定。 TSA.1的2-和5-苯基取代基几乎垂直于环己基环，导致轨道重叠降低并降低推定的过渡状态。种系抗体的活性位点似乎具有增加程度的柔性-CDRH3在TSA.1的结合时从活性位点向外移动4.9。我们建议在种系抗体中的这种构象灵活性，这导致对TSA.1的较低的结合亲和力允许沿反应坐标沿2-苯基取代基的二偏角角的动态变化。这些构象变化又导致增强的眶倍数和增加的催化率。这些研究表明，蛋白质和衬底动力学在该抗体催化反应中发挥着关键作用。

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