  Hybrid quantum mechanical-molecular mechanical (QM/MM) simulations are widely
used in studies of enzymatic catalysis. Until recently, it has been cost
prohibitive to determine the asymptotic limit of key energetic and structural
properties with respect to increasingly large QM regions. Leveraging recent
advances in electronic structure efficiency and accuracy, we investigate
catalytic properties in catechol O-methyltransferase, a representative example
of a methyltransferase critical to human health. Using QM regions ranging in
size from reactants-only (64 atoms) to nearly one-third of the entire protein
(940 atoms), we show that properties such as the activation energy approach
within chemical accuracy of the large-QM asymptotic limits rather slowly,
requiring approximately 500-600 atoms if the QM residues are chosen simply by
distance from the substrate. This slow approach to asymptotic limit is due to
charge transfer from protein residues to the reacting substrates. Our large
QM/MM calculations enable identification of charge separation for fragments in
the transition state as a key component of enzymatic methyl transfer rate
enhancement. We introduce charge shift analysis that reveals the minimum number
of protein residues (ca. 11-16 residues or 200-300 atoms for COMT) needed for
quantitative agreement with large-QM simulations. The identified residues are
not those that would be typically selected using criteria such as chemical
intuition or proximity. These results provide a recipe for a more careful
determination of QM region sizes in future QM/MM studies of enzymes.
