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 https://icekat.herokuapp.com/icekat.

中文翻译：

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ICEKAT：一种交互式在线工具，用于根据连续酶动力学轨迹计算初始速率。

背景技术连续酶动力学分析通常用于高通量应用，因为与不连续分析相比，它们允许快速获取大量动力学数据并增加置信度。然而，数据分析在高通量酶测定中通常是限速的，因为手动检查和从单个动力学轨迹中选择线性范围很麻烦，并且容易出现用户错误和偏差。当前可用的软件程序专门用于分析复杂的酶模型。尽管广泛使用初始速率测定来处理动力学数据集，但不存在用于从连续酶动力学轨迹快速分析初始速率的简单和自动化程序。结果 交互式连续酶动力学分析工具 (ICEKAT) 被开发用于半自动计算连续酶动力学轨迹的初始速率，特别适用于评估 Michaelis-Menten 和 EC50/IC50 动力学参数，以及高- 通量筛选分析。ICEKAT 允许用户使用方便的基于浏览器的选择工具以交互方式拟合动力学轨迹，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。作为一个测试案例，我们快速分析了超过 500 条连续的酶动力学曲线，这些曲线来源于关于蛋白质赖氨酸脱乙酰酶 SIRT1 对小分子激活剂反应的实验数据。结论 ICEKAT 允许同时可视化各个初始速率拟合和由此产生的 Michaelis-Menten 或 EC50/IC50 动力学模型拟合，以及来自高通量筛选分析的命中。除了为执业酶学家提供方便的程序外，ICEKAT 还是一个有用的教学辅助工具，可以实时直观地演示不正确的初始速率拟合如何影响计算的 Michaelis-Menten 或 EC50/IC50 动力学参数。为方便研究社区，我们已在 https://icekat.herokuapp.com/icekat 在线免费提供 ICEKAT。ICEKAT 也是一种有用的教学辅助工具，可以实时直观地演示不正确的初始速率拟合如何影响计算的 Michaelis-Menten 或 EC50/IC50 动力学参数。为方便研究社区，我们已在 https://icekat.herokuapp.com/icekat 在线免费提供 ICEKAT。ICEKAT 也是一种有用的教学辅助工具，可以实时直观地演示不正确的初始速率拟合如何影响计算的 Michaelis-Menten 或 EC50/IC50 动力学参数。为方便研究社区，我们已在 https://icekat.herokuapp.com/icekat 在线免费提供 ICEKAT。