Characterization On Mechanism Of Metallo-?-lactamase Binding Zn(?) And Their Activities In Bacteria By ITC

?-lactam antibiotics is one of the most important drugs in the treatment of bacterial infections.The overuse of ?-lactam antibiotics in clinical,agricultural and animal husbandry has led to the production of a large number of drug-resistant bacteria.One of the most important mechanisms of bacterial resistance is the production of metal?-lactamases?M?Ls?to hydrolyze the ?-lactam ring of ?-lactam antibiotics and make them inactive.Nearly all known ?-lactam are hydrolyzed by M?Ls,but there are no enzyme inhibitors available for clinical use.In order to deal with the drug resistance of bacteria,scientists have been studying the structure and catalytic mechanism of M?Ls.Based on this idea,we first expressed and purified the three subgroups representative enzymes CcrA,NDM-1,VIM-2,IMP-1,ImiS and L1,and characterized them by SDS-PAGE and UV-Vis,respectively.On the basis of the purified enzymes,the demetallized apo-M?Ls proteins were prepared.The activity of apo-NDM-1 and recombinant NDM-1 were characterized.In order to further study the structure and catalytic mechanism of M?Ls,the interaction of M?Ls with Zn²⁺ was characterized by isothermal titration calorimeter?ITC?for the first time.The modes of Zn²⁺ binding to M?Ls in three subgroups were determined,and a series of thermodynamic parameters were obtained,including stoichiometry?N?,equilibrium constant?Kd?,entropy chang??S?,enthalpy change??H?.On the basis of ITC,CcrA and NDM-1 enzymes of mononuclear and dinuclear species were prepared and their catalytic activities were determined.A ITC method was developed to monitor and inhibit the real-time activity of M?Ls in living bacteria.The inhibitory effects of enzyme inhibitors EDTA,D-captoril,azolylthioacetamide and ebselen on M?Ls-producing bacteria were evaluated.Furthermore,the thermal effect of cefazolin,a antibiotic hydrolyzed by different drug-resistant bacteria,was studied,and important scientific data and conclusions were obtained.The ITC technique was developed in this paper.It provides a new way to study the structure and function of metallic enzymes in complex biological network environment.