Two central steps in the hydrolysis of lactam antibiotics catalyzed by mononuclear metallo-beta-lactamases, formation of the tetrahedral intermediate and its breakdown by proton transfer, are studied for model systems using the density functional B3LYP method. Metallo-beta-lactamases have two metal ion binding sites, one of which is occupied in the mononuclear species. In this work it is assumed that catalysis takes place at zinc site 1, which is modeled by the metal ion, three imidazole rings, and a hydroxide ion. The lactam ring, a minimal model of beta-lactam antibiotics, is initially coordinating to the zinc ion. Potential proton shuttles from the second (unoccupied) metal-binding site (water, Asp, or Cys) are included in some calculations. The calculated reaction barrier for formation of the tetrahedral intermediate is 13 kcal/mol, close to what is observed experimentally for the rate-limiting step. The barrier for the breakdown of the intermediate is low, 0-10 kcal/mol, if it is assisted by a water molecule or by a Cys or Asp model. Thus, the results indicate that proton transfer is not rate-limiting, and that any of the residues from the second metal site may function as proton shuttle. For most studied systems, the tetrahedral structure is a stable intermediate. Moreover, the C-N bond in the lactam ring is intact in this intermediate, as well as in the following transition state-its cleavage is induced by proton transfer to the nitrogen atom in the lactam ring. However, for the model with Asp as a proton shuttle, attack of the zinc-bond hydroxide ion seems to be concerted with the proton transfer. We have also studied the effect of replacing one of the histidine ligands by an asparagine or glutamine residue, giving a zinc site representative of other subclasses of metallo-beta-lactamases. This has only a small effect on the calculated reaction barriers. Likewise, if the zinc ion is replaced by cadmium, only small changes in the reaction barrier for proton transfer are seen, whereas the barrier for the formation of the tetrahedral intermediate increases by 3 kcal/mol and the intermediate is destabilized by 5 kcal/mol.
Journal of Physical Chemistry B, 2003, Vol 107, Issue 10, p. 2366-2375