Mechanistic studies on B.cereus metallo-beta-lactamase BcII (conferencista invitado)

ALEJANDRO J, VILA

Eretria, Grecia

Congreso; 10th Beta-Lactamase Meeting; 2008

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; text-align:justify; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> 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 b-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.