Metal content and localization in the catalytically relevant forms of Co(II)-substituted metallo-beta-lactamase BcII

LLARRULL, L.I.; TIONI, M.F.; PERIYANNAN, G. R.; BENNETT, B.; VILA, A.J.

Montevideo

Conferencia; 6th International Conference of Biological Physics, 5th Southern Cone Biophysical Congress, XXXVI Reunion Anual Sociedad Argentina de Biofisica; 2007

The metallo-b-lactamase (MbL) BcII from Bacillus cereus was first crystallized with only one Zn(II) ion tetrahedrally coordinated to three His residues and a bridging H2O/OH- molecule (3H-site). Spectroscopic experiments and a second crystal structure later indicated that BcII was also capable of binding a second metal ion, which is trigonal bipyramidally coordinated to Asp120, Cys221, His263, the bridging H2O/OH- and an additional water molecule (DCH-site). We recently carried out a study on Co(II) uptake by BcII which indicated that three equivalents of Co(II) are bound sequentially, with macroscopic binding constants KD1 = 0.12 ± 0.06 uM, KD2 = 0.16 ± 0.04 uM and KD3 = 94 uM. Besides, an exchange of the metal ion between the 3H and the DCH-sites occurs on mono-Co(II) BcII. The identity of the catalytically relevant species of BcII and the mechanism of action of this enzyme are still matter of debate. In this work we studied penicillin G hydrolysis catalyzed by Co(II)-BcII through stopped-flow rapid-scanning experiments and Rapid Freeze Quench-EPR experiments, at different Co(II)/BcII ratios. Our results indicate that both the mono-Co(II) and di-Co(II) forms of BcII are active, and that penicillin G hydrolysis proceeds by way of a branched kinetic pathway that involves Co(II) dissociation from the dinuclear enzyme-substrate complex to give rise to a mononuclear enzyme-substrate adduct. This model implies that mono-Co(II) BcII is active, in contrast to the previously reported conclusions drawn from kinetic experiments carried out in the presence of excess Co(II), which suggested that di-Co(II) BcII was the only active species. Our data also indicated that binding of penicillin G to mono-Co(II) BcII gives rise to an enzyme-substrate complex with the metal ion localized in the DCH site. This proposal contrasts to the generally accepted hypothesis that the mononuclear active species would bear a single metal ion bound to the 3H site.