...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants
【24h】

Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants

机译：实时对酶动力学的非稳态分析阐明了底物关联和解离速率：通过分析萤火虫荧光素酶突变体的示范

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

Firefly luciferase has been widely used in biotechnology and biophotonics due to photon emission during enzymatic activity. In the past, the effect of amino acid substitutions (mutants) on the enzymatic activity of firefly luciferase has been characterized by the Michaelis constant, K-M. The K-M is obtained by plotting the maximum relative luminescence units (RLU) detected for several concentrations of the substrate (luciferin or luciferyl-adenylate). The maximum RLU is used because the assay begins to violate the quasi-steady state approximation when RLU decays as a function of time. However, mutations also affect the time to reach and decay from the maximum RLU. These effects are not captured when calculating the K-M. To understand changes in the RLU kinetics of firefly luciferase mutants, we used a Michaelis-Menten model with the non-steady state approximation. In this model, we do not assume that the amount of enzyme-substrate complex is at equilibrium throughout the course of the experiment. We found that one of the two mutants analyzed in this study decreases not only the dissociation rate (k(off)) but also the association rate (k(on)) of luciferyl-adenylate, suggesting the narrowing of the structural pocket containing the catalytic amino acids. Furthermore, comparative analysis of the nearly complete oxidation of luciferyl-adenylate with wild-type and mutant firefly luciferase reveals that the total amount of photons emitted with the mutant is 50-fold larger than that with the wild type, on average. These two results together indicate that the slow supply of luciferyl-adenylate to the enzyme increases the total number of photons emitted from the substrate, luciferyl-adenylate. Analysis with the non-steady state approximation model is generally applicable when enzymatic production kinetics are monitored in real time.
机译：由于在酶活性期间，由于光子发射，萤火虫荧光素酶已广泛用于生物技术和生物体源性。在过去，氨基酸取代（突变体）对萤火虫荧光素酶酶活性的影响已经表征了Michaelis常数K-M。通过绘制检测到几种浓度的基质（Luciferin或Luciferyl-腺苷酸）来绘制检测到的最大相对发光单元（Rlu）获得K-M。使用最大RLU，因为当RLU衰减作为时间的函数时，测定开始违反准稳态近似。然而，突变也会影响到达最大RLU的时间和衰减的时间。计算K-M时不会捕获这些效果。为了了解萤火虫荧光素酶突变体的RLU动力学的变化，我们使用了具有非稳态近似的Michaelis-Menten模型。在该模型中，我们不认为酶 - 衬底复合物的量在实验过程中在平衡处处于平衡状态。我们发现本研究中分析的两个突变体中的一个不仅降低了解离率（K（off）），而且还降低了荧光纤维 - 腺苷酸的缔合率（K（ON）），表明含有催化剂的结构口袋氨基酸。此外，与野生型和突变萤火虫荧光素酶几乎完全氧化的比较分析显示，突变体发出的光子的总量比野生型为50倍。这两种结果在一起表明荧光素-亚苯基酯与酶的缓慢供应增加了从基材，荧光纤维 - 腺苷酸释放的光子的总数。随着非稳态近似模型的分析通常适用于实时监测酶促生产动力学。

著录项

  • 来源
    《Biochemistry》 |2019年第23期|共8页
  • 作者

    Dale Renee; Ohmuro-Matsuyama Yuki; Ueda Hiroshi; Kato Naohiro;

  • 作者单位

    Louisiana State Univ Dept Biol Sci Baton Rouge LA 70803 USA;

    Tokyo Inst Technol Inst Innovat Res Lab Chem &

    Life Sci Nagatsuta Cho Yokohama Kanagawa Japan;

    Tokyo Inst Technol Inst Innovat Res Lab Chem &

    Life Sci Nagatsuta Cho Yokohama Kanagawa Japan;

    Louisiana State Univ Dept Biol Sci Baton Rouge LA 70803 USA;

  • 收录信息
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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