| Abstract
| - Hybrid Car−Parrinello QM/MM calculations are used to investigate the reaction mechanism ofhydrolysis of a common β-lactam substrate (cefotaxime) by the monozinc β-lactamase from Bacillus cereus(BcII). The calculations suggest a fundamental role for an active site water in the catalytic mechanism.This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordinationnumber and providing a proton donor adequately oriented for the second step. The free energy barriers ofthe two reaction steps are similar and consistent with the available experimental data. The conservedhydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes toorient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to thezinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energybarrier, which is consistent with the stability of cefotaxime in water solution. The modeled Michaeliscomplexes for other substrates are also characterized by the presence of an ordered water molecule inthe same position, suggesting that this mechanism might be general for the hydrolysis of different β-lactamsubstrates.
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