Hybrid Car-Parrinello QM/MM calcns. are used to investigate the reaction mechanism of hydrolysis of a common b-lactam substrate (cefotaxime) by the monozinc b-lactamase from Bacillus cereus (BcII). The calcns. suggest a fundamental role for an active site water in the catalytic mechanism. This water mol. binds the zinc ion in the first step of the reaction, expanding the zinc coordination no. and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available exptl. data. The conserved hydrogen bond network in the active site, defined by Asp-120, Cys-221, and His-263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water mol. to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water soln. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water mol. in the same position, suggesting that this mechanism might be general for the hydrolysis of different b-lactam substrates. [on SciFinder (R)]