Due to its importance in the pharmaceutical industry, ligand dynamic simulations have experienced a great expansion. Using all-atom models and cutting-edge hardware, one can perform nonbiased ligand migration, active site search, and binding studies. In this Letter, we demonstrate (and validate by PCR mutagenesis) how these techniques, when combined with quantum mechanics, open new possibilities in enzyme engineering. We provide a complete analysis where (1) biophysical simulations produce ligand diffusion and (2) biochemical modeling samples the chemical event. Using such broad analysis, we engineer a highly stable peroxidase activating the enzyme for new substrate oxidation after rational mutation of two nonconserved surface residues. In particular, we create a new surface-binding site, quantitatively predicting the in vitro change in oxidation rate obtained by mutagenic PCR and achieving a comparable specificity constant to active peroxidases.

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