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 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.