Study On The Interaction Between Some Enzymes And Gallic Acid,Chlorogenic Acid,vanillic Acid

Research about the interaction mechanism between biomacromolecules and forest active small medicinal molecules has become a frontier and hot topic in the field of forestry science,natural product science,pharmacy and biology science subjects.The investigation of interaction between forest active medicinal molecules with protein is not only beneficial to further understand the structure and function of biomacromoleculars and forest active drugs to provide theoretical guidance for expanding the application scope of forest products,but also is helpful for us to understand the mechanism of drugs transferring and to exploit forest active drugs which would be more effective to diseases and have targeting.In this paper,we use spectroscopy experiment combined with molecular docking and molecular dynamics simulation technique,respectively exploring the interaction mechanism of the forest active drugs gallic acid(GA)and chlorogenic acid(CGA)with lysozyme(LYS),exploring the interaction mechanism of the forest active drug vanillic acid(VA)and polyphenol oxidase(PPO)and constructing the VA-PPO three-dimensional interaction fingerprint spectrum by spectrum experiment,providing a reference to establish the standard fingerprint spectrum.The main content of this thesis includes the following parts:1.Properties of LYS and PPO were briefly introduced,research advances methods of interaction between biological macromolecules and small molecules and the principles of interaction though spectroscopic analysis were present,the research content and significance of this thesis were summarized.2.The interaction mechanisms between GA and LYS have been investigated and compared by spectroscopic techniques in combination with molecular dynamics(MD)simulations.The free binding energies were calculated using poission-boltzmann surface area method.The results from fluorescence spectroscopy indicated that GA could bind to LYS to form static complex.The apparent binding constants are different due to the different temperatures.The binding of GA-LYS mainly depended on hydrogen bonding and Van der Waals' forces.The interaction between the protein LYS residue and the GA molecule(2.12 nm),which indicated the efficiency of energy transfer according to F?rster theory.GA changed the hydrophobicity of the binding area and the conformation3.transformation of the GA binding with LYS was a process of two-state model.Molecular dynamics simulation was performed to reveal that GA could bind to LYS mainly by hydrophobic interaction and hydrogen bonding.The result of root mean square deviation(RMSD)confirmed that GA could bind with LYS to form a stable complex and the root mean square fluctuation(RMSF)results could display the stability of GA-LYS complex at 298 K was higher than that at 310 K.The lower the temperature,the smaller the protein flexibility,the more stable the binding between LYS and GA,and the results of the experiment results are consistent with the spectra.The calculated free binding energies from MM-PBSA/GBSA method showed hydrophobic interaction and electrostatic interaction were the predominant intermolecular forces stabilizing the complex.The computational simulations showed consistent results with spectral experiment.4.The mechanism of combining LYS with CGA had been studied.The binding parameters were detected by spectrum experiment under physiological conditions,and the molecular modeling techniques had been used to investigate the interaction mechanism between CGA and LYS.Molecular docking revealed that CGA bind to LYS mainly by van der Waals forces and hydrogen bonds interactions,and there is a hydrophobic interaction.The results from spectroscopy indicated that the static binding exited between CGA and LYS with significant bond strong.The value of binding distances r<7nm,which described them comply with non-radiative energy transfer theory.The conformational pattern of CGA-LYS shows “two-state” model.Molecular dynamics simulation was performed to reveal that CGA could bind to LYS mainly by hydrophobic interaction and hydrogen bonding.The result of RMSD confirmed that CGA could bind with LYS to form a stable complex and RMSF results could display the stability of CGA-LYS complex at 298 K was higher than that at 310 K.The higher the temperature,the bigger the protein flexibility,the less stable the binding between LYS and GA,and the results of the experiment results are consistent with the spectra.The calculated free binding energies from MM-PBSA/GBSA method showed electrostatic interaction and hydrophobic interaction were the predominant intermolecular forces stabilizing the complex.5.The interaction mechanisms were explored by using fluorescence spectroscopy/UV-visible absorbance spectroscopy in combination with molecular modeling studies.The three-dimensional fingerprint of the VA-PPO system was built.The fluorescence spectroscopy indicated that the interaction between PPO and VA resulted in fluorescence enhancement.The thermodynamic6.parameters suggested that the hydrophobic interaction predominantly stabilized the complex.The values of r(2.48 nm)was lower than 7 nm after interaction between protein PPO and the VA molecule,which indicated the efficiency of energy transfer according to F?rster theory.The VA-PPO ?-UV-F fingerprint was built by using the spectroscopic data,which revealed the interaction of the active molecule VA binding with PPO.Molecular modeling revealed one major hydrophobic interaction and six hydrogen bond interactions between VA and the residues Met258,His88,His109,His240,His244 and His274 of PPO.The computational simulations showed consistent results with spectral experiment.This work also built a three-dimensional fingerprint of the VA-PPO system.