Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. 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. The presented ITC-dependent workflow was used to determine these parameters for the cofactor-dependent ketoreductase Gre2p under different reaction conditions. Using a standardized approach with the implementation of sample quality control by DLS, we obtained high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. The results revealed that this enzyme operates with an ordered sequential mechanism in which the cofactor NADPH binds first, which then allows binding of the substrate
NDK. In addition, the enzyme was found to be affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility, a mandatory prerequisite to achieve robust modeling, is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. ... mehrrepository. Because they can be automated and scaled for high-throughput, the combination of different approaches such as the one presented will provide the high-quality data needed for the development of enzymes and biocatalytic processes through machine learning to accelerate the future development of industrial biocatalysis.