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

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

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY

AMER CHEMICAL SOC

Año: 2008 vol. 130 p. 15852 - 15863

0002-7863

Metallo--lactamases hydrolyze most -lactam antibiotics. The lack of a successful inhibitor forthem 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 usedto characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able tohydrolyze imipenem in both 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 -lactambond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonantstructure 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, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-statebased inhibitors for all three classes of metallo-beta-lactamases.