The experimentally determined Michaelis constant Kmc results from a combination of two effects: the recognition of the substrate by the enzyme and the molecular interactions between substrate and solvent. By separating substrate recognition from solvent effects, the thermodynamic activity-based Michaelis constant Kma allows for an unambiguous comparison of how different substrates fit into the substrate binding site. Kma of a poorly water-soluble substrate is calculated from the experimentally determined concentration-based Michaelis constant Kmc and its activity coefficient at infinite dilution γ∞. Comparing the Kma of different substrates instead of the experimentally determined Kmc prevents misinterpretations of the molecular basis of enzyme-substrate interactions. While n-octane showed the lowest Kmc value of six P450BM-3 substrates, its Kma was 500 fold higher than aniline, indicating that the binding of n-octane is mainly driven by its low water solubility, while binding of aniline is driven by its shape complementarity. For three substrates (aniline, oct-1-yne, n-octane), γ∞ was reliably calculated by molecular dynamics simulations, either in binary substrate-water mixtures or in ternary mixtures including DMSO as cosolvent. It is demonstrated that the widely used DMSO has a considerable effect on the measured Kmc value. Depending on the substrate, addition of 10% v/v DMSO increases Kmc by up to a factor of 11. To make biocatalytic experiments reproducible, it is therefore of utmost importance to carefully report the reaction conditions. The reliable simulation of activity coefficients in complex mixtures allows for an unambiguous comparison of enzyme-substrate interactions and provides a predictive tool for the design of biocatalytic processes.
Keywords: Activity coefficient; Aniline; Cosolvent; Molecular dynamics simulation; Oct-1-yne; P450(BM-3); Thermodynamic activity-based Michaelis constant; n-Octane.
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