The Molecular Recognition And Interaction Between TEM-1 ?-Lactamase And Three ?-Lactam Antibiotics

Drug-resistance of antibiotics is one of the key social issues during human medicinal behaviors. This issue is possible to be addressed by several strategies including rational use of antibiotics, development of new antibiotics and investigation on the interaction between antibiotics and their in vivo specific targets. As largely used antibiotics, the reason for the drug-resistance of ?-lactam antibiotics is believed to be:the generation of drug-resistance target protein (?-lactamase) by ampicillin resistance Escherichia coli; the catalysis and hydrolysis of ?-lactam antibiotics by the enzyme. TEM-1 is the most largely known and classic ?-lactamase. The process of the enzyme catalyzing and hydrolyzing P-lactam antibiotics involves multi-steps, among which the recognition and binding of the antibiotics is found to be the first one.This dissertation researches the recognition between TEM-1 and three antibiotics by site-directed molecular docking firstly, selecting penicillin G, cephalexin and cefoxitin as probes. Then we further sdudy the mechanism of interaction between TEM-1 and the three antibiotics by spectrometry and affinity chromatography. It is capable of elucidating the mechanism of the binding of ?-lactam antibiotics to TEM-1 in molecular level. It is also possible to play particularly important role in addressing the issue of drug-resistance of antibiotics and developing new antibiotics with high efficacy. The content of this dissertation consists of 3 chapters where the main contribution of the author is listed as below: 1. The bindings of penicillin G, cephalexin and cefoxitin to TEM-1 were simulated by molecular docking. Then the spectral method was used to verification TEM-1 conformation changes when TEM-1 binded with three antibiotics. It was confirmed that molecular recognition was the key factor for the interaction between TEM-1 and the three antibiotics. The results of ultraviolet absorption spectrum and fluorescence spectrum confirmed that the conformation change of TEM-1 was the key factor for the interaction between TEM-1 and the three antibiotics. The results showed that TEM-1 molecules had the capacity to form hydrogen bonds with the three antibiotics. The numbers of the bonds were six for penicillin G, five for cephalexin and three for cefoxitin. Depended upon these hydrogen bonds, a new complexation was produced between TEM-1 and the three antibiotics with the ratio of 1:1, respectively. The interaction between TEM-1 and the three antibiotics was mainly driven by hydrogen bonds and Van de Waals'forces. The investigation by ultraviolet absorption spectrum showed that the intensity positively corresponded to the concentration of the antibiotics; the peak profile presented a red shift trend. The results of quenching study with the use of KI indicated that the quenching constant of the complexation was greater than that of TEM-1. Moreover, the number of amino residuals serving as chromophore was decreased after the formation of the complexation. Synchronous fluorescence spectrum showed that the fluorescence intensity was decreased along with the growing concentrations of the three antibiotics when ??. were 60 nm and 15 nm, respectively. Second order derivative spectrophotometric study showed that both TEM-1 and its inclusion complexation with the three antibiotics have two negative peaks when the emission wave length was 278 nm. Furthermore, the peak intensity and the situation have also changed. Only one negative peak with the loss of the intensity was found when the emission wave length was 295 nm.2. Fluorescence spectrum and affinity chromatography was used to sdudy the mechanism of interaction between TEM-1 and the three antibiotics. The results showed that TEM-1 bound to the three antibiotics at the ratio of 1:1. The order of the association constants were penicillin G> cephalexin> cefoxitin. The quenching effect of the three beta-lactam antibiotic on TEM-1 belonged to static quenching. The thermodynamic parameters including enthalpy change(?H), entropy change (?S) and free energy change were all less than zero, providing a proof that the binding between the three antibiotics and TEM-1 was a spontaneous exothermic reaction. Hydrogen bond and van der waals were the main force during the interaction. In addition, the apparent activation energy was calculated to be negative which indicated that there is no energy barrier along the reaction direction, which enable the reaction to proceed spontaneous. By the energy transfer theory of Forster, the binding distance (r) of the three antibiotics to TEM-1 were found to be less than 7 nm and situated in the range of 0.5 Ro< r < 1.5 Ro. This result demonstrated that the energy transfer during the binding of the three antibiotics to TEM-1 were non-radiative. The results of affinity chromatography showed that the immobilized TEM-1 still has the bioactivity of recognizing the three antibiotics. The interaction between them was in accorded with Langmuir model. The association constants of penicillin G, cephalexin and cefoxitin were determined to be 3.54×104 M-1,1.89×104 M-1 and 1.23×104 M-1 by frontal analysis, giving an affinity order of penicillin G> cephalexin> cefoxitin. This order was in good line with the result by fluorescence spectrum, which confirmed the reliability of the application of the immobilized TEM-1 in analyzing the binding of antibiotics to the enzyme.