Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry
Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry
复制标题
用等温滴定量热法建立可重复酶模型
DOI：
10.1021/acscatal.1c02076
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发表时间：
2021-05
期刊：
影响因子：
12.9
通讯作者：
F. Ott;Kersten S. Rabe;C. Niemeyer;G. Gygli
中科院分区：
文献类型：
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作者：
F. Ott;Kersten S. Rabe;C. Niemeyer;G. Gygli
为了在技术过程中应用酶，需要详细了解分子机制。酶催化的动力学和热力学参数对于更有效地规划、建模和实施生物催化过程至关重要。虽然动力学参数Km和kcat通常可以通过光学方法获得，但热力学参数的测定需要更复杂的方法。等温滴定量热法（ITC）允许对酶反应催化循环中各个步骤的动力学和热力学参数进行无标记和高灵敏度的分析。然而，由于ITC易受由于酶的变性或附聚引起的干扰，因此必须始终考虑酶样品的均匀性，并且这可以通过动态光散射（DLS）分析来实现。我们在这里报告使用ITC依赖的工作流程，以确定一个辅因子依赖的酶的动力学和热力学数据。使用标准化的方法，通过DLS实施样品质量控制，我们获得了适用于酶反应机制高级建模的高质量数据。具体而言，我们研究了立体选择性反应催化的NADPH依赖性酮还原酶Gre2p在不同的反应条件下。结果表明，该酶的操作与有序的顺序机制，并受到底物或产物抑制取决于反应缓冲液。通过指定标准操作程序、使用编程工作流程进行数据分析并将所有数据存储在F.A.I.R.中，确保数据重现性。（可查找、可访问、可互操作和可重用）存储库（https：//doi.org/10.15490/fairdomhub.1.investigation.464.1）。我们的工作突出了这种复杂的多底物反应的结合和动力学研究的效用。
To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, Km and kcat, are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.