Reaction Mechanism and Catalytic Determinants of a Highly Active De Novo Metalloesterase

MID1sc10 is an engineered zinc esterase that was evolved
to high
activity (kcat/Km ∼ 106 M–1·s–1) and enantiospecificity over ten rounds of mutagenesis
and screening. Although its evolutionary trajectory had stagnated,
there is potentially room for further augmenting this enzyme’s
catalytic efficiency. As atomistic details could provide valuable
insights for developing an even more efficient enzyme, we used quantum
mechanics/molecular mechanics calculations to characterize MID1sc10’s
reaction mechanism. Our data suggest that the reaction occurs via
a two-step process, involving metal ion-mediated ester cleavage followed
by regeneration of the zinc coordination site. The initial step is
rate-limiting, with a Gibbs energy barrier of 15.1 kcal·mol–1, in good agreement with the experimental value (kcat = 1.64 s–1, ΔG‡ = 17.3 kcal·mol–1). Additionally, detailed atomistic and energetic information was
used to retrospectively rationalize how the introduction or removal
of charged residues during directed evolution optimized MID1sc10.
Applying the same method to predict mutations to increase the efficiency
of MID1sc10 suggested that mutating Asp25, Glu26, Asp74, or Glu81
to neutral polar or positively charged residues, or replacing Val62
or Gln66 with negatively charged residues, would help stabilize the
rate-limiting transition state.