Mechanistic studies on B.cereus metallo-beta-lactamase BcII

A. J. VILA

Eretria

Encuentro; 10th beta Lactamase Meeting; 2008

Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopy were used to characterize the reaction of Co(II)-BcII with imipenem. The kinetic data recorded under non-steady state conditions, together with the RFQ-EPR experiments, show that Co(II)-BcII is able to hydrolyze imipenem both in the mono- and dinuclear forms. The species that accumulates upon imipenem hydrolysis is an enzyme-intermediate adduct where the beta lactam bond has already been cleaved. This intermediate is a metal-bound anionic species, with a resonant structure, that is stabilized by the metal ion at the DCH or Zn2 site. This species exhibits a very intense absorption band at 407 nm and Resonance Raman spectroscopy revealed a feature at 1541 cm-1 that corresponds to a distorted C-N or C-C double bond. This represents a novel, previously unforeseen intermediate, which is related to the specific chemical nature of carbapenems. Since carbapenems are the only substrates in common for B1, B2 and B3 lactamases, which all share a DCH or Zn2 site with a similar role in catalysis, the identification of this intermediate could be exploited as a first step towards the design of transition state based inhibitors for all three classes of metallo beta lactamases.