Molecular dynamics derived life times of active substrate binding poses explain K M of laccase mutants.

Fungal laccases (EC 1.10.3.2) 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 K M , but a molecular understanding of this effect is missing. We explored the effect of TvL mutations on K M 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 K M due to mutation consistently correlate with the dynamics of the substrates within the substrate-binding site. We find that K M 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 K M values. Our results indicate that hydrophobic packing of the substrate near the T1 binding site of the laccase is instrumental for high turnover via K M . We also address the more general question of how enzymes such as laccases gain advantage of lower K M despite the Sabatier principle, which disfavors a stable enzyme-substrate complex. Our data suggest that the observed K M 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.