Reference : Positively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 beta-Lactamase...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/28817
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
English
Jacquin, Olivier mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines, Centre d'Ingénierie des Protéines > >]
Balbeur, Dorothée mailto [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]
Damblon, Christian mailto [Université de Liège - ULg > Département de chimie (sciences) > Chimie biologique structurale >]
Marchot, Pierre mailto [Université de Liège - ULg > Département de chimie appliquée > Génie chimique - Systèmes polyphasiques >]
De Pauw, Edwin mailto [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]
Roberts, Gordon C K [University of Leicester > Department of Biochemistry > Henry Wellcome Laboratories of Structural Biology > > >]
Frère, Jean-Marie mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
Matagne, André mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines, Centre d'Ingénierie des Protéines > >]
2009
Journal of Molecular Biology
Academic Press
392
5
1278-1291
Yes (verified by ORBi)
International
0022-2836
1089-8638
London
United Kingdom
[en] metallo-beta-lactamases ; cooperativity ; NMR ; mass spectrometry ; circular dichroism
[en] 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 H 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 >= 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. (C) 2009 Elsevier Ltd. All rights reserved.
http://hdl.handle.net/2268/28817
10.1016/j.jmb.2009.07.092

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