Hybrid QM/MM and DFT investigations of the catalytic mechanism and inhibition of the dinuclear zinc metallo-beta-lactamase CcrA from Bacteroides fragilis.

Based on hybrid QM/MM molecular dynamics simulation and density functional theoretical (DFT) calculations, we investigate the mechanistic and energetic features of the catalytic action of dizinc metallo-beta-lactamase CcrA from Bacteroides fragilis. The 200 ps QM/MM simulation of the CcrA enzyme in complex with nitrocefin shows that the substrate beta-lactam moiety is directed toward the active site dizinc center through the interactions of aminocarbonyl and carboxylate groups with the two active site zinc ions and the two conserved residues, Lys167 and Asn176. From the determination of the potential energy profile of a relevant enzymatic reaction model, it is found that the nucleophilic displacement reaction step proceeds with a low-barrier height, leading to the formation of an energetically favored reaction intermediate. The results also show that the high catalytic activity of the CcrA enzyme stems from a simultaneous operation of three catalytic components: activation of the bridging hydroxide nucleophile by zinc-coordinated Asp86; polarization of the substrate aminocarbonyl group by the first zinc ion; stabilization of the negative charge developed on the departing amide nitrogen by the second zinc ion. Consistent with the previous experimental finding that the proton-transfer reaction step is rate-limiting, the activation energy of the second step is found to be 1.6 kcal/mol higher than that of the first step. Finally, through an examination of the structural and energetic features of binding of a thiazolidinecarboxylic acid inhibitor to the active site dizinc center, a two-step inhibition mechanism involving a protonation-induced ligand exchange reaction is proposed for the inhibitory action of a tight-binding inhibitor possessing a thiol group.