Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Abstract

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, K m and k cat , 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.

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

“…Since all aspects lead to a lack of experimental reproducibility, insufficient standardization and inconsistencies in data management are particularly troubling and hamper further development of the field [4d,5,28] . To improve reproducibility and credibility of experimental studies, various efforts to implement guidelines, such as the National Research Data Infrastructure Consortium (NFDI, https://nfdi.de), [29] are recently being applied in biocatalysis research [30] …”

“…[4d,5,28] To improve reproducibility and credibility of experimental studies, various efforts to implement guidelines, such as the National Research Data Infrastructure Consortium (NFDI, https://nfdi.de), [29] are recently being applied in biocatalysis research. [30]…”

Isothermal Titration Calorimetry in Biocatalysis

“…The wide field involving ITC to study protein-protein or protein-ligand (metal ion, inhibitor, cofactor) interactions, thermodynamics of membrane proteins, as well as the study of protein stability are excluded from this review, but excellent reviews can be found elsewhere (Freire et al, 1990;Jelesarov and Bosshard, 1999;Ward and Holdgate, 2001;Holdgate, 2001;O'Brien and Haq, 2004;Grossoehme et al, 2010;Draczkowski, et al, 2014;Rajarathnam and Rösgen, 2014;Quinn, et al, 2016). A number of (tutorial) reviews on the study of enzyme kinetics using ITC are already available (Todd and Gomez, 2001;O'Brien and Haq, 2004;Bianconi, 2007;Demarse et al, 2013;Quinn and Hansen, 2019;Wang, et al, 2020;Ott, et al, 2021). The focus of this minireview is to cover developments in enzyme calorimetry that are relevant for the biocatalysis field.…”

Orthogonal protein decoration of DNA nanostructures based on SpyCatcher–SpyTag interaction

“…Indeed, an induced fit mechanism has been proposed for Gre2p, which leads to a conformational change of the active site upon NADPH binding. 17 This process could be decelerated by immobilizing Gre2p on DON. Additionally, the high negative charge density on DON as well as tight packing on beads could lead to a changed local environment and thus result in unfavorable interactions between Gre2p and DON.…”