Metallo-�-lactamases (MBL's) are enzymes that hydrolyze and inactivate �-lactam ring containing antibiotics. MBL's are classified into three subclasses (B1-B3), depending upon their amino acid homologies and biochemical properties. The MBL's bind 1-2 Zn(II) ions and are not susceptible to classical �-lactamase inhibitors, such as clavulanate and sulbactam. Our overall goal is to elucidate the catalytic mechanisms of several representative MBL's and identify common mechanistic aspects that could be targeted for inhibitors. Previous studies have revealed that the metal center plays a crucial role in the hydrolysis reaction and that the invariant loop may �clamp down� on the substrate promoting catalysis. In our studies, we have used spectroscopic studies, such as UV-visible, NMR, EPR, and EXAFS spectroscopies to probe the metal binding sites, and rapid freeze quench (RFQ) spectroscopic studies to probe the metal binding sites during catalysis. RFQ-EXAFS studies showed that the metal ions perform different roles in catalysis with the metal ion in the Zn1 site playing a major role in intermediate formation and the metal ion in the Zn2 playing a major role in product formation. CW- and RFQ-DEER studies have been used to probe the dynamics of the invariant loop during catalysis. The RFQ-DEER studies demonstrate that the loop moves 8-10 � away from the metal binding site during intermediate formation (10 ms after mixing) then back to the resting position in the product complex. Future studies will involve probing the reactions at faster times (<1 ms).

Probing the active site and mobile loop of metallo-�-lactamases during catalysis

246th National Meeting (2013)

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