Abstract (Expand)
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.
Authors: Felix Ott, Kersten S. Rabe, Christof M. Niemeyer, Gudrun Gygli
Date Published: 3rd Sep 2021
Publication Type: Journal
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