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Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Ott, Felix ORCID iD icon 1; Rabe, Kersten S. ORCID iD icon 1; Niemeyer, Christof M. ORCID iD icon 1; Gygli, Gudrun 1
1 Institut für Biologische Grenzflächen (IBG), Karlsruher Institut für Technologie (KIT)


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. ... mehr

Verlagsausgabe §
DOI: 10.5445/IR/1000137212
Veröffentlicht am 06.09.2021
DOI: 10.1021/acscatal.1c02076
Zitationen: 3
Web of Science
Zitationen: 3
Zitationen: 6
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Biologische Grenzflächen (IBG)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2021
Sprache Englisch
Identifikator ISSN: 2155-5435, 2155-5435
KITopen-ID: 1000137212
HGF-Programm 43.33.11 (POF IV, LK 01) Adaptive and Bioinstructive Materials Systems
Erschienen in ACS catalysis
Verlag American Chemical Society (ACS)
Band 11
Heft 17
Seiten 10695–10704
Vorab online veröffentlicht am 12.08.2021
Schlagwörter (S)-stereoselective ketoreductase; kinetic model; FAIR/F.A.I.R.; reproducibility; quality control; dynamic light scattering; UV−vis spectrophotometry
Nachgewiesen in Dimensions
Web of Science
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