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|Title:||The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase|
Karsisiotis, Andreas Ioannis
Roberts, Gordon C. K.
|Publisher:||American Society for Biochemistry and Molecular Biology|
|Citation:||Journal of Biological Chemistry, 2016, 291 (31), pp. 16124-16137|
|Abstract:||Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG0) of 32 ± 2 kJ·mol−1. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG0 value of 65 ± 1.4 kJ·mol−1. These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.|
|Rights:||Copyright © 2016 The American Society for Biochemistry and Molecular Biology, Inc. Deposited with reference to the publisher’s open access archiving policy.|
|Appears in Collections:||Published Articles, Dept. of Biochemistry|
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|J. Biol. Chem.-2016-Montagner-16124-37.pdf||Published (publisher PDF)||2.53 MB||Adobe PDF||View/Open|
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