Exploring the role and the binding affinity of a second zinc equivalent in B. cereus metallo-beta-lactamase.

RASIA, R M; VILA, A J

BIOCHEMISTRY

AMER CHEMICAL SOC

Año: 2002 vol. 41 p. 1853 - 1860

0006-2960

Metallo-beta-lactamases are a newly characterized family of zinc enzymes present in several pathogenic strains that represent an emerging clinical threat. Enzymes from different organisms exhibit an outstanding functional diversity, particularly in the metal ion requirements for activity. We have investigated the effect of the second zinc(II) equivalent in the enzyme âLII from Bacillus cereus, naturally active in the mono-zinc(II) form. The enzyme is reversibly inactivated at low pH, due to dissociation of the two zinc(II) equivalents. The pH profile indicates that zinc-bound water in the mono-zinc(II) enzyme possesses a pKa below 4.9, indicating that a second zinc(II) equivalent is not needed for nucleophile activation. Instead, the second zinc(II) may contribute to properly anchor Asp120, that ultimately orients the attacking nucleophile in binuclear enzymes. This role may be fulfilled by Arg121 in mono-zinc enzymes, as suggested by the kinetic study of the R121C mutant in betaLII. In addition, it is demonstrated that Arg121 is not responsible for the low binding affinity of betaLII toward a second zinc(II) equivalent.