ICEKAT: an interactive online tool for calculating initial rates from continuous enzyme kinetic traces
ICEKAT: an interactive online tool for calculating initial rates from continuous enzyme kinetic traces
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DOI：
10.1186/s12859-020-3513-y
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发表时间：
2020-05-14
影响因子：
3
通讯作者：
Smith, Brian C.
中科院分区：
文献类型：
--
作者：
Olp, Michael D.;Kalous, Kelsey S.;Smith, Brian C.
背景连续酶动力学测定通常用于高通量应用中，因为与不连续测定相比，它们允许快速采集大量动力学数据并增加置信度。然而，在高通量酶测定中，数据分析通常是限速的，因为手动检查和从单个动力学迹线选择线性范围是繁琐的，并且容易出现用户错误和偏差。目前可用的软件程序是专门设计用于分析复杂的酶模型。尽管广泛使用的初始速率测定处理动力学数据集，没有简单的和自动化的程序存在的快速分析的初始速率从连续的酶动力学轨迹。结果开发了一个交互式连续酶动力学分析工具（ICEKAT），用于从连续酶动力学轨迹半自动计算初始速率，特别适用于Michaelis-Menten和EC 50/IC 50动力学参数的评估，以及高通量筛选试验的结果。ICEKAT允许用户使用方便的基于浏览器的选择工具交互式地拟合动力学轨迹，改善了定义范围以适应Microsoft Excel和Graphpad Prism等通用程序所涉及的繁琐步骤，同时仍然保持确定初始速率的简单性。作为测试案例，我们快速分析了500多个连续酶动力学轨迹，这些轨迹来自蛋白质赖氨酸脱乙酰酶SIRT 1对小分子激活剂反应的实验数据。结论ICEKAT允许同时可视化个体初始速率拟合和所得Michaelis-Menten或EC 50/IC 50动力学模型拟合，以及来自高通量筛选测定的命中。除了作为一个方便的程序，实践酶学，ICEKAT也是一个有用的教学辅助工具，实时直观地演示如何不正确的初始速率拟合可以影响计算的Michaelis-Menten或EC 50/IC 50动力学参数。为了研究社区的方便，我们在网上免费提供ICEKAT。
Background Continuous enzyme kinetic assays are often used in high-throughput applications, as they allow rapid acquisition of large amounts of kinetic data and increased confidence compared to discontinuous assays. However, data analysis is often rate-limiting in high-throughput enzyme assays, as manual inspection and selection of a linear range from individual kinetic traces is cumbersome and prone to user error and bias. Currently available software programs are specialized and designed for the analysis of complex enzymatic models. Despite the widespread use of initial rate determination for processing kinetic data sets, no simple and automated program existed for rapid analysis of initial rates from continuous enzyme kinetic traces. Results An Interactive Continuous Enzyme Kinetics Analysis Tool (ICEKAT) was developed for semi-automated calculation of initial rates from continuous enzyme kinetic traces with particular application to the evaluation of Michaelis-Menten and EC50/IC50 kinetic parameters, as well as the results of high-throughput screening assays. ICEKAT allows users to interactively fit kinetic traces using convenient browser-based selection tools, ameliorating tedious steps involved in defining ranges to fit in general purpose programs like Microsoft Excel and Graphpad Prism, while still maintaining simplicity in determining initial rates. As a test case, we quickly analyzed over 500 continuous enzyme kinetic traces resulting from experimental data on the response of the protein lysine deacetylase SIRT1 to small-molecule activators. Conclusions ICEKAT allows simultaneous visualization of individual initial rate fits and the resulting Michaelis-Menten or EC50/IC50 kinetic model fits, as well as hits from high-throughput screening assays. In addition to serving as a convenient program for practicing enzymologists, ICEKAT is also a useful teaching aid to visually demonstrate in real-time how incorrect initial rate fits can affect calculated Michaelis-Menten or EC50/IC50 kinetic parameters. For the convenience of the research community, we have made ICEKAT freely available online at .