Depicting the Catalytic Mechanism of the Metallo-beta-lactamase of B.cereus

LLARRULL, L.I.; VILA, A.J.

Bariloche

Congreso; XXXIX Annual Meeting of SAIB, XXXII Annuall Meeting of SAB and Bariloche Protein Symposium; 2003

SAB - SAIB

Metallo-beta-lactamases are enzymes with a zinc binding motif in their active site, that is essential for their hydrolytic activity towards different antibiotics. A rational design of new inhibitors should necessarily rely on a thorough knowledge of their catalytic mechanism, still unveiled. Asp90 is conserved in all known metallo-beta-lactamases. This residue binds zinc in enzymes containing two metal ions, whereas it is involved in a strong hydrogen bond interaction with the nucleophilic OH- in mono zinc enzymes. Asp90 might participate in the mechanism of mono zinc enzymes, as is the case of B.cereus BcII, either: (1) by defining the appropriate orientation of the attacking nucleophile, or (2) through acid/base catalysis. To analyse the mechanism of the mono and bi-metallic BcII, we evaluated the solvent kinetic isotope effect (KIE) on the hydrolysis of different substrates. The differences observed in solvent KIE of these two forms of the enzyme can be interpreted in terms of different rate determining steps. In the mono zinc enzyme, the rate determining step would be the nucleophilic attack of the Zn-bound OH- to the beta-lactam ring, whereas in the bi zinc enzyme, a proton transfer step woud be rate-limiting. Solvent KIE experiments carried out on the mutants D90N and D90S suggest that Asp90 does not have an acid/base role in catalysis.