• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Thermodynamic and structural basis for transition-state stabilization in antibody-catalyzed hydrolysis.
【24h】

Thermodynamic and structural basis for transition-state stabilization in antibody-catalyzed hydrolysis.

机译：抗体催化水解中过渡态稳定的热力学和结构基础。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, k(cat)/k(uncat)=895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56 and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 A resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an "induced fit" manner, especially at theL1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive DeltaS, differing from general thermodynamic characteristics of antigen-antibody interactions. This positive DeltaS could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in DeltaG between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger k(cat) value of 6D9. Interestingly, the DeltaDeltaG was mainly because of the DeltaDeltaH. The correlation between k(cat) and DeltaDeltaH is suggestive of "enthalpic strain" leading to destabilization of antibody-substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9.
机译：通过用半抗原过渡态类似物（TSA）免疫诱导诱导的催化抗体6D9和9C10催化非生物活性氯霉素单酯衍生物的水解，从而产生生物活性氯霉素。这些抗体严格按照以下理论关系来稳定过渡态以催化水解反应：对于6D9，k（cat）/ k（uncat）= 895，K（S）/ K（TSA）= 900，对于9C10， k（cat）/ k（uncat）= 56，K（S）/ K（TSA）= 60。为了阐明抗体催化反应的分子基础，确定了6D9的晶体结构，并使用等温滴定量热法分析了6D9和9C10与底物和TSA的结合热力学。以2.25 A的分辨率测定未配体的6D9 Fab的晶体结构，并将其与先前报道的TSA配体的6D9 Fab的晶体结构进行比较，表明TSA以“诱导的契合”方式结合到抗原结合位点的疏水口袋中。方式，尤其是在L1和H3 CDR环上。热力学分析表明6D9以阳性DeltaS结合TSA的底物，这与抗原-抗体相互作用的一般热力学特征不同。这种正的DeltaS可能是由于6D9与底物或Trp H100i介导的TSA之间的疏水相互作用。底物和结合6D9的TSA之间的DeltaG差异大于结合9C10的差异，这与更大的6D9的k（cat）值具有良好的相关性。有趣的是，DeltaDeltaG主要是因为DeltaDeltaH。 k（cat）与DeltaDeltaH之间的相关性提示“焓菌株”导致抗体-底物复合物的不稳定。结合X射线结构分析，热力学分析表明，结合底物后，抗体将底物中酯部分的构象从平面Z形式改变为热力学不稳定的扭曲构象，然后转变为过渡态。焓应变还通过使基态不稳定来促进过渡态的稳定，而对于更有效的催化抗体6D9，它的程度要大得多。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号