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 enzyme catalysis are crucial to plan, model, implement biocatalytic processes more efficiently. While kinetic parameters, Km kcat, often accessible by optical methods, determination requires sophisticated methods. Isothermal titration calorimetry (ITC) allows label-free highly sensitive analysis individual steps catalytic cycle an reaction. However, since ITC susceptible interferences due denaturation or agglomeration enzymes, homogeneity sample must always be considered, this can accomplished means dynamic light scattering (DLS) analysis. We here report on use ITC-dependent work flow determine both data for cofactor-dependent enzyme. Using standardized approach with implementation quality control DLS, we obtain high-quality suitable advanced modeling reaction mechanism. Specifically, investigated stereoselective reactions catalyzed NADPH-dependent ketoreductase Gre2p under different conditions. The results revealed that operates ordered sequential mechanism affected substrate product inhibition depending buffer. Data reproducibility ensured specifying standard operating procedures, using programmed workflows analysis, storing all F.A.I.R. (findable, accessible, interoperable, reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our highlights utility combined binding studies such complex multisubstrate reactions.