Trapping and characterization of a reaction intermediate in carbapenem hydrolysis by B. cereus metallo-beta-lactamase.

MARIANA FLORENCIA TIONI; LETICIA IRENE LLARRULL; ANDRÉS A. POEYLAUT-PALENA; MARCELO MARTÍ; MIGUEL SAGGU; GOPAL R. PERIYANNAN; ERNESTO G. MATA; BRIAN BENNETT; DANIEL H. MURGIDA; ALEJANDRO J. VILA

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY

American Chemical Society

Año: 2008 vol. 130 p. 15852 - 15863

0002-7863

 Metallo-beta-lactamases hydrolyze most beta-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 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 beta-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, 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-state based inhibitors for all three classes of metallo-beta-lactamases.