Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo-β-lactamase

The hydrolysis of the beta-lactam compound nitrocefin by the metallo-beta-lactamase from B. cereus (BcII) was studied by pre-steady state kinetics measurements, followed by absorbance, fluorescence and diode array detection. In contrast with the results reported for the homologous enzymes CcrA from B. fragilis and L1 from S. maltophilia, no accumulation of an anionic intermediate could be evidenced. The rationale for this observation can be tracked on the lower binding affinity toward a second Zn(II) ion in this enzyme, and the modification of a flexible active site loop, that might contribute to stabilize the anionic intermediate. Analysis of the substrate binding and product formation rates does not support a recently proposed mechanistic scheme that contemplates a non-negligible accumulation of this intermediate in nitrocefin hydrolysis by BcII.