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首页> 外文期刊>Journal of Molecular Biology >Structural basis of the transition-state stabilization in antibody-catalyzed hydrolysis.
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Structural basis of the transition-state stabilization in antibody-catalyzed hydrolysis.

机译：抗体催化水解中过渡态稳定的结构基础。

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The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis; the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic-aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate.
机译：产生针对过渡态类似物（TSA）的催化抗体6D9催化非生物活性氯霉素单酯的水解生成氯霉素。已经表明6D9在催化中利用了结合亲和力。 TSA相对于底物的差异亲和力等于速率增强。为了揭示6D9对TSA和底物的识别机制，我们对6D9的Fv片段（6D9-Fv）进行了NMR分析，以及定点诱变和停流动力学分析。在六个6D9-Fv突变体中，Y58（H）A和W100i（H）A与TSA的亲和力显着降低，而其底物结合亲和力与野生型6D9-Fv相同。停止流动力学研究表明，TSA与6D9-Fv的结合是通过诱导拟合机制发生的。相反，对于Y58（H）A和W100i（H）A，未观察到诱导拟合的TSA结合机制。从NMR实验中，我们确定了具有化学位移的残基，这些残基受到配体结合的干扰。受TSA结合影响的残基位于X射线研究确定的TSA结合位点，以及远离结合位点的区域。另一方面，受底物结合影响的残基位于TSA结合位点。对于W100i（H）A，除了结合位点以外的残基均不受配体结合的影响。根据这些结果和晶体结构，我们得出结论，TSA结合诱导构象变化，涉及形成芳族-芳族相互作用和氢键。这些相互作用可以解释相对于底物对TSA的不同亲和力。 W100i（H）可能在诱导构象变化中起重要作用。目前的NMR研究使我们能够可视化酶催化中过渡态稳定化的概念，其中过渡态接触比底物更好。

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