| The latest superbugs can accrurately catalyze the hydrolysis process of the beta-lactam ring structure in beta-lactam antibiotics by producing their target protein metallo-beta-lactamases, and make clinical use of almost all beta-lactam antibiotics ineffective. Up to now, the molecular recognition and interaction between metallo-beta-lactamase molecules and beta-lactam antibiotic molecules have not been elucidated for people.The Bc Ⅱ from Bacillus cereus is a representative metallo-beta-lactamase. In this dissertation, combined method of investigation of the binding processes of Bc Ⅱ with Penicillin V (PV) and Sulbactam (Sul) was carried out. Spectroscopic techniques (fluorscence spectrum, ultraviolet spectrum and circular dichroism spectrum), theoretical prediction methods (molecular docking and dynamic simulation), and determination of thermodynamic parameters were adopted. The molecular recognition and interaction of Bc Ⅱ with antibiotic were investigated though the observation of the changes of Bc Ⅱ conformation, the space distribution and orientation of the related amino acid residues in Bc Ⅱ, and the related functional groups in PV and Sul, the intermolecular distance, the number of the interactional sites, and the type of interactional force between Bc Ⅱ and antibiotics molecules (PV and Sul).1. The enzymatic assay of Bc Ⅱ with PV was studied under pH7.0Tris-HCl buffer solution at277K and298K. The enzyme activity at277K was38.1%of that at298K. The acid product of the enzymatic reaction started occurrence at about two mins. There were mutual recognition and mutual influence between Bc Ⅱ and PV in this period of the binding process. On the basis of studies on UV spectra and fluorescence emission spectra of Bc Ⅱ with PV, it was confirmed that the ratio of PV and Bc Ⅱ in saturation binding process at277K was0.67. The molecular recognition between Bc Ⅱ and PV in the binding process at277K for two mins was studied by spectroscopic techniques (circular dichroism spectra、synchronous fluorescence spectra、fluorescence quenching by KI spectra and two derivative spectra of the fluorescence emission spectra). It was found that there were slight changes of Bc Ⅱ conformation, the decreased content of the ordered structure a-helix (30.4%to28.9%), the increased content of the random structure (34.2%to36.0%), and the weak changes of chromophoric amino acid residues on the surface of Bc Ⅱ. These changes of Bc Ⅱ were in order to adapt to binding with PV and prepare for the cleavage process of beta-lactam ring of PV. The interactions between Bc Ⅱ and PV were studied by fluorescence emission spectra under pH7.0Tris-HCl buffer solution at277K,281K and285K. It was found that non-fluorescence static complex formed between Bc Ⅱ and PV, and the number of binding sites of Bc Ⅱ were1.03-1.05, and the binding constant of the complex BcⅡ-PV were1.15-2.00×106L/mol. Thermodynamic parameters were calculated and their interaction force were revealed. Negative entropy (ΔH°=-45.38kJ/mol), negative enthalpy (ΔS°=-42.80J/mol) and negative Gibbs free energy indicated that hydrogen bonds and Van der Waals interactions played a major role in stabilizing the complex BcⅡ-PV, and the interaction of Bc Ⅱ and PV was a spontaneous process. The kinetics simulation of the interaction between Bc Ⅱ and PV revealed that there was a stable intermediate in the binding process of Bc Ⅱ-PV. According to the Forster non-radiative energy transfer theory, the binding distance between Bc Ⅱ and PV was2.02nm. These results were substantiated by molecular docking studies.2. The enzymatic assay of Bc Ⅱ with Sul was studied under pH7.0Tris-HCl buffer solution at277K and298K. The enzyme activity at277K was25.0%of that at298K. The acid product of the enzymatic reaction started occurrence at about two mins. There were mutual recognition and mutual influence between Bc Ⅱ and Sul in this period of the binding process. On the basis of studies on UV spectra and fluorescence emission spectra of Bc Ⅱ and Sul, it was confirmed that the ratio of Sul and Bc Ⅱ in saturation binding process at277K was0.53. The molecular recognition of Bc Ⅱ and Sul in the binding process at277K for two mins was studied by spectroscopic techniques (circular dichroism spectra, synchronous fluorescence spectra, fluorescence quenching by K.I spectra and two derivative spectra of the fluorescence emission spectra). It was found that there were slight changes of Bc Ⅱ conformation, the decreased content of the ordered structure a-helix (30.5%to29.0%), the increased content of the random structure (34.1%to35.9%), and the weak changes of chromophoric amino acid residues on the surface of Bc Ⅱ. These changes of Bc Ⅱ were in order to adapt to binding with Sul and prepare for the chemical reaction of beta-lactam ring of Sul cleavage process. The interaction of Bc Ⅱ and Sul was studied by fluorescence emission spectra under pH7.0Tris-HCl buffer solution at277K,281K and285K. It was found that non-fluorescence static complex formed between Bc Ⅱ and Sul, and the number of binding sites of Bc Ⅱ were0.89-0.93, and the binding constant of the complex Bc Ⅱ-Sul were1.49-3.98×105L/mol. Thermodynamic parameters were calculated and their interaction force were revealed. Negative entropy(ΔH°=-80.70kJ/mol), negative enthalpy(ΔS°=-184.17J/mol) and negative Gibbs free energy indicated that hydrogen bonds and Van der Waals interactions played a major role in stabilizing the complex, and the interaction of Bc Ⅱ and Sul was a spontaneous process.The kinetics simulation of the interaction between Bc Ⅱ and Sul revealed that there was a stable intermediate in the binding process of Bc Ⅱ-Sul. According to the Forster non-radiative energy transfer theory, the binding distance between Bc Ⅱ and Sul was2.14nm. These results were substantiated by molecular docking studies.3. The molecular recognition and interaction of the two binding system were compared separately from the assay of enzyme activity at low temperature, the measurement of fluorscence spectrum, ultraviolet spectrum and circular dichroism spectrum, and the determination of thermodynamic parameters and kinetic calculation. It was concluded that the conformation of Bc Ⅱ had weak changes in these two binding process, the stable fluorescence static quenching complex of Bc Ⅱ-PV and Bc Ⅱ-Sul were formed in these two binding processes. In addtion, it were confirmed that the main international force of the two systems (Bc Ⅱ-PV and Bc Ⅱ-Sul) were both hydrogen bonds and Van der Waals because of negative enthalpy and negative entropy, the binding process of two system were both exothermic reaction and spontaneous process because of negative enthalpy and negative Gibbs free energy, and then there were both a stable intermediate in the binding process of two system. Based on the differences of the molecular structure of these two antibiotics, there were some differences of two system on the binding distance, the affinity, and the ability of the antibiotics binding to Bc Ⅱ. Although the active site of Bc Ⅱ in two interactional process were identical, the side chain of PV was out of active site of Bc Ⅱ, by contrast, the whole Sul was in active site of Bc Ⅱ because of Sul’s small steric stabilization. It was concluded by molecular docking studies that there were three groups of hydrogen bonding between Bc Ⅱ and PV, including the group of hydrogen bonding in the side of PV, while there were two groups of hydrogen bonding between Bc Ⅱ and Sul. These differences resulted in that the affinity of Bc Ⅱ to PV and the stability of complex Bc Ⅱ-PV were both higher than that of Bc Ⅱ-Sul binding system. The fluorescence quenching constants and the binding constant of two binding systen also corroborated these above inferences.Above analysis explored the essential characteristics, the molecular recognition and interaction mechanism of Bc Ⅱ with PV and Sul in their binding process. Taking this one step further, these studies from the level of molecules might provide some valuable information for further research and development of new and high-performance superantibiotics to the latest superbugs. |
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