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

LLARRULL, L.I.; DAL PERARO, M.; CARLONI, P.; VILA, A.J.

Ventura, California

Conferencia; Gordon Research Conferences Graduate Research Seminars: Bioinorganic Chemistry; 2004

Gordon Research Conferences

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. The kinetic study of BcII mutant variants in which Asp90 was modified for aminoacids with different chemical characterisits show that Asp90, is essential for full activity. The kinetics studies were complemented with spectroscopic studies on the Co(II) substituted enzymes and with molecular dynamic calculations. These studies indicate changes in the coordination sphere in the mutants proteins which could correlate with the decreased activity. The MD studies show that an Asp at position 90 define the appropriate orientation of the attacking nucleophile, and hence would give rise to improved hydrolytic acyivity. Stopped-flow studies on the WT enzyme indicate that Zn(II) is also required for substrate binding to BcII.