Fungal laccases (EC 18.104.22.168) are important multi-copper oxidases with broad substrate specificity. Laccases from Trametes versicolor (TvL) are among the best-characterized of these enzymes. Mutations in the substrate-binding site of TvL substantially affect KM, but a molecular understanding of this effect is missing. We explored the effect of TvL mutations on KM for the standard laccase substrate 2,6-dimethoxyphenol using 4500 ns of molecular dynamics, docking, and MMGBSA free energy computations. We show that changes in KM due to mutation consistently correlate with the dynamics of the substrates within the substrate-binding site. We find that KM depends on the lifetime (“dynamic stability”) of the enzyme-substrate complex as commonly assumed. We then further show that MMGBSA-derived free energies of substrate binding in the active pose consistently reproduce large vs. small experimental KM values. Our results indicate that hydrophobic packing of the substrate near the T1 binding site of the laccase is instrumental for high turnover via KM. We also address the more general question of how enzymes such as laccases gain advantage of lower KM despite the Sabatier principle, which disfavors a stable enzyme–substrate complex. Our data suggest that the observed KM relates directly to the lifetime of the active substrate pose within a protein. In contrast, the thermochemical stability of the enzyme–substrate complex reflects an ensemble average of all enzyme–substrate binding poses. This distinction may explain how enzymes work by favoring longer residence time in the active pose without too favorable general enzyme–substrate interactions, a principle that may aid the rational design of enzymes.