...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Toward the Elucidation of the Catalytic Mechanism of the Mono-ADP-Ribosyltransferase Activity of Pseudomonas aeruginosa Exotoxin A
【24h】

Toward the Elucidation of the Catalytic Mechanism of the Mono-ADP-Ribosyltransferase Activity of Pseudomonas aeruginosa Exotoxin A

机译：阐明铜绿假单胞菌外毒素A的单ADP核糖基转移酶活性的催化机理

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

The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses.The rate constants for binding of the NAD~+ substrate to the enzyme were fond to be 4.7(+-)0.4muM~(-1)s~(-1) and 194(+-)15s~(-1) for k_(on) and k_(off),respectively.The k_(on) and k_(off) rate constants for the eEF-2 substrate binding to the enzyme were 320(+-)39muM~(-1) s~(-1) and 131(+-)22s~(-1),respectively.A potent,competivie inhibitor against the enzyme,1,8-naphthalimide,bound the enzyme with k_(on) and k_(off) rates of 82(+-)9muM~(-1)s~(-1)and 51(+-)6s~(-1),respectively.Furthermore,the binding on and off rates for the reaction products,ADP-ribose and nicotinamide,were too rapid for detection with the stopped-flow technique.Investigation of the presteady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle.Binding of NAD~+ and smaller compounds representing the vraious parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence.The binding dtata revealed a significant structural change in the enzyme upon NAD~+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD~+ constituents.Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose,and the results indicate that the reaction ivolves a random-order ternary complex mechanism.Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme,and fluorescence resonace energy transfer measurements indicated that whear germ eEF-2 is oligomeric in solution.
机译：通过稳态和停流动力学分析研究了铜绿假单胞菌外毒素A的单ADP-核糖基转移酶活性的催化机理.NAD〜+底物与酶的结合速率常数为4.7（+ -）0.4μM〜（-1）s〜（-1）和194（+-）15s〜（-1）分别用于k_（on）和k_（off）.k_（on）和k_（off） eEF-2底物与酶结合的速率常数分别为320（+-）39muM〜（-1）s〜（-1）和131（+-）22s〜（-1）。 1,8-萘二甲酰亚胺以82（+-）9muM〜（-1）s〜（-1）和51（+-）6s〜的k_（on）和k_（off）比率​​结合酶（-1）。此外，反应产物ADP-核糖和烟酰胺的结合开/关速度太快，无法用停流技术检测.ADP-核糖的稳态前动力学研究毒素酶的转移酶活性表明在催化循环中没有形成前稳态复合物。通过内在毒素荧光的荧光猝灭研究了NAD〜+和代表该底物各个部分的较小化合物的表达。结合数据表明，NAD〜+结合后酶的结构发生了重大变化，这是无法解释的。用烟酰胺和eEF-2-ADP-核糖进行了产物抑制研究，结果表明该反应是一个随机的三元复合机理。详细的动力学分析表明，eEF-2底物在酶的作用下呈S形动力学行为，荧光共振能量转移测量表明，麦芽胚芽eEF-2在溶液中是低聚物。

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