Catalytic mechanism of class B2 metallo-β-lactamase

Abstract

The initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-β-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/ molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be ∼14 kcal/ mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the Oδ2-Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-β-lactamase. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.