Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

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.

若要将酶应用于工业工艺，需对其分子机制具备详尽认知。酶催化反应的动力学与热力学参数，对于更高效地规划、建模及实施生物催化工艺至关重要。尽管动力学参数米氏常数（Km）与催化常数（kcat）通常可通过光学方法获取，但热力学参数的测定则需要更为精密的方法。等温滴定量热法（Isothermal titration calorimetry, ITC）可实现无标记且高灵敏度的分析，用于测定酶反应催化循环中各步骤的动力学与热力学参数。然而，由于酶的变性或聚集会对ITC造成干扰，因此必须始终考量酶样品的均一性，这可通过动态光散射（Dynamic light scattering, DLS）分析来实现。本研究报道了一种依托ITC的工作流，用于测定辅因子依赖型酶的动力学与热力学数据。通过采用标准化方法，并结合DLS进行样品质量管控，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，我们研究了不同反应条件下，依赖NADPH的酮还原酶Gre2p所催化的立体选择性反应。结果显示，该酶以有序序列机理运作，且其活性会受底物或产物抑制的影响，具体取决于反应缓冲液的组成。通过制定标准操作流程、采用程序化数据分析工作流，并将所有数据存储于可发现、可访问、可互操作、可复用（F.A.I.R., findable, accessible, interoperable, and reusable）知识库中（https://doi.org/10.15490/fairdomhub.1.investigation.464.1），确保了数据的可重复性。本研究彰显了针对此类复杂多底物反应开展结合与动力学联合研究的应用价值。