Structure of the First-Shell Active Site in Metallolactamase: Effect of Water Ligands

In this study we have examined the behavior of the first-shell active site of metallolactamases as a function of water both bound directly as a zinc ligand and hydrogen bonded to protein-residue ligands in the active site and as a function of metal−metal distance. The inherent effective interaction energy of the bimetallic metallolactamase active site is relatively flat between the metal cations. This leaves the metal−metal distance susceptible to both perturbations from environmental interactions and protonation of active-site residues. Although the crystal structure of the active site of the zinc lactamase from Bacteroides fragilis is the initial starting point for the structure optimizations, structures very different from the equilibrium crystal structure as well as details of the water hydrogen-bonding pattern in the active site are obtained. Structures with Zn−Zn distances >4 Å, with each metal cation acting as the center of a separate complex, result when only the crystallographic water that is directly coordinated to the Zn is included in the representation of the active site. When more crystallographic waters are included, the structure remains essentially unchanged from the crystal structure. In addition, a class of structures with even shorter metal−metal distances is calculated with the active site cysteine and the hydroxide ion bound to both zinc cations. This class of conformation is low in energy and includes several hydrogen bonds between the active-site residues and surrounding waters. Protonation of the active site also yields a metal−metal distance either >4 Å or comparable to that in the crystal structure, depending on whether the proton is added to the bridging hydroxide or to the carboxylate of the aspartate 103 ligand. The Zn−O distances of an X-ray structure obtained at a pH of 5.6 agree with those of the active site protonated at the hydroxide. The long-distance structure is the lowest in energy for the Zn−Zn enzyme; however, when zinc is substituted by cadmium, the long-distance structure becomes higher in energy because the cadmium cation does not polarize the ligated water as strongly as zinc. The lowest-energy structure for the Zn−Cd system is predicted to have the zinc bound at the site with three histidine ligands, in agreement with a recent experimental deduction. We suggest that structures that differ from the crystal structure can play a role in the reaction or in the initial stages of metal binding, as indicated by the UV−visible spectrum observed in the cobalt-substituted enzyme. Water that is hydrogen bonded to the active site is also believed to play an important role in the catalytic mechanism; one of the waters may function as the nucleophile.