Trapping and Characterization of a Reaction Intermediate in Imipenem Hydrolysis by B. cereus Metallo-â-lactamase

TIONI, M. F.; LLARRULL, L. I.; POEYLAUT-PALENA, A. A.; MARTÍ, M. A.; SAGGU, M.; PERIYANNAN, G. R.; MATA, E. G.; BENNETT, B.; MURGIDA, D. H.; VILA, A. J.

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY

ACS

Lugar: Washington; Año: 2008 vol. 130 p. 15852 - 15863

0002-7863

Metallo-β-lactamases hydrolyze most β-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate.Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem both in the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the β-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species, with a novel resonant structure, that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate, that is related to the chemical nature of carbapenems. Since carbapenems are the only substrates cleaved by B1, B2 and B3 lactamases, 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-β-lactamases.