A Lysine-Targeted Affinity Label for Serine β-Lactamase Also Covalently Modifies New Delhi Metallo-β-Lactamase-1 (NDM-1)

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-β-lactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 μM) and kinact (0.045 min–1) values. Mass spectrom...