An insight into the role of Asp120 in the catalytic mechanism of the metallo-beta lactamase of B. cereus

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

Angra dos Reis, RJ

Congreso; 1st Latin American Protein Society Meeting; 2004

Metallo-beta-lactamases (MBLs) 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 is a zinc ligand in enzymes containing two metal ions, whereas in mono zinc enzymes it is involved in a strong hydrogen bond interaction with the nucleophilic OH-. It has been predicted that Asp90 is essential in the mechanism of mono zinc enzymes, as is the case of the MBL from B.cereus (BcII), either: (1) by defining the appropriate orientation of the attacking nucleophile, or (2) through acid/base catalysis. To evaluate the role of Asp90 in the hydrolysis of beta-lactam antibiotics, we engineered four mutants of the enzyme BcII in this position. All of the mutants showed decreased activity against nitrocefin; with the following trend: D90E > D90N, D90Q > D90S. None of the mutants was totally inactive, as expected in terms of the proposed mechanism. Characterization of the structure of the active sites by means of UV-VIS spectroscopy on the cobalt substituted derivatives indicated that a tetrahedral metal site similar to the one in wild type BcII is conserved in these mutants. MD simulations were carried out on the mono zinc mutant enzymes, by constraining the metal site in a tetrahedral geometry, according to the spectroscopic data. The results of these simulations are consistent with the catalytic efficiencies displayed by each mutant. These results as a whole suggest that Asp90 participates in the hydrogen bond network which orients the water/OH- molecule, which is a ligand of the tetrahedral zinc site in monoZn BcII. The orientation of the nucleophile modulates the enzyme catalytic efficiency. The Asp90 mutants present the same solvent kinetic isotope effect observed in the wild type enzyme, which indicates that the replacement of this residue does not alter significantly the mechanism of catalysis. These results are not consistent with a rate-determining acid/base role of Asp90 in catalysis.