Positively cooperative binding of zinc ions to Bacillus cereus 569/H/9 beta-lactamase II suggests that the binuclear enzyme is the only relevant form for catalysis.
Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K(1)/K(2)> or =5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K(2)<80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility.
Jacquin
Olivier
O
Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie B6, 4000 Liège (Sart Tilman), Belgium.
Balbeur
Dorothée
D
Damblon
Christian
C
Marchot
Pierre
P
De Pauw
Edwin
E
Roberts
Gordon C K
GC
Frère
Jean-Marie
JM
Matagne
André
A
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011485:Protein Binding; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D047090:beta-Lactams; C455979:beta-lactamase BcII; D001618:beta-Lactamases; D015032:Zinc