Interpretation of cytochrome P450 monooxygenase kinetics by modeling of thermodynamic activity

The experimentally determined Michaelis constant K_m^c 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 K_m^a allows for an unambiguous comparison of how different substrates fit into the substrate binding site. K_m^a of a poorly water-soluble substrate is calculated from the experimentally determined concentration-based Michaelis constant K_m^c and its activity coefficient at infinite dilution γ^∞. Comparing the K_m^a of different substrates instead of the experimentally determined K_m^c prevents misinterpretations of the molecular basis of enzyme-substrate interactions. While n-octane showed the lowest K_m^c value of six P450_BM-3 substrates, its K_m^a 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 K_m^c value. Depending on the substrate, addition of 10% v/v DMSO increases K_m^c 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.