The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase*

Metallo-β-lactamases catalyse 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 (∆G°) of 32 ± 2 kJ·mol^−1 . For holoBcII, a first noncooperative 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. 2D 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 ∆G° 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 in order to maintain enzymatic activity.