| Our interest lies in a motif appearing in a number of hydrolytic metalloenzyme active sites; the “bridged binuclear metal” motif. It consists of two metal cations (usually divalent) bridged by an endogenous ligand (usually a carboxylate, coordinating the metals in a bidentate fashion), an exogenous one (a water molecule, a substrate, hydroxide) or both. In most of these enzymes, the motif has been found to be critical for catalysis. Aeromonas Proteolytica Aminopeptidase (AAP) is a well studied enzyme, with a relatively simple and a quasi symmetrical active site that can be used as a paradigm to study these hydrolytic metalloenzyme active sites and the reactions they catalyze. The native form of AAP contains two Zn2+ cations in the motif. We briefly present a crystallographic structure of the Co2+ substituted enzyme to see how it compares to the native form. We start to investigate the role of the “bridged binuclear metal” motif by simple and general (non-enzyme specific) ab-initio (DFT) models where Mg2+ is our metal of choice. Subsequently, an aminopeptidase substrate analog is added to the motif to describe a prototype amide hydrolysis reaction with Zn2+ and Mg2+ as the metals. Their respective coordination spheres are completed by terminal water ligands. Finally, we attempt to model the AAP amide hydrolysis reaction. Again, an aminopeptidase substrate analog is added to the motif which contains Zn2+ or Mg2+ in the metal sites. The metal coordination spheres are completed by carboxylate and N-containing ligands mimicking the choice of ligands in the AAP active site. |
