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.