Binding of Zinc(II) to Apo‐Metallo‐β‐Lactamases: Characterization by Isothermal Titration Calorimetry

Antibiotic resistance caused by metallo‐β‐lactamases (MβLs) is a global public health threat. In this work, we first directly investigated the binding of Zn 2+ to apo‐MβLs through isothermal titration calorimetry. It was found that carbapenem and cephamycin resistance metallo‐β‐lactamase (CcrA), New Delhi metallo‐β‐lactamase‐1 (NDM‐1) and Stenotrophomonas maltophilia Metallo‐β‐lactamase‐1(L1) bind Zn 2+ in a sequential mode, while imipenemase‐1 (IMP‐1) and Verona Integron‐encoded metallo‐β‐lactamase‐2 (VIM‐2) bind Zn 2+ in a mode of varying levels of cooperativity, in which the mononuclear species was not detectable, and also, the mode was verified through the titration of Co 2+ into apo‐VIM‐2 monitored by UV–vis. The thermodynamic and kinetic parameters revealed the binding of Zn 2+ to the active sites was independent of stoichiometry. Analysis of the parameters determined suggested that the binding of B1 and B3 MβLs to Zn 2+ depends on the binding affinity and/or Gibbs free energy change of Zn 1 and Zn 2 sites of the enzymes, and two metal sites have almost equal contributions for full activity of the MβLs. The information gained in this work is helpful for the further mechanism studies and the development of the broad‐spectrum MβL inhibitors.