Molecular Dynamics Simulation Studies On The Adenylate Kinase Catalytic Cycle And The Substrate Recognition Mechanism Of Lipase PLA₁

Protein structure and function are closely related,and structural changes on protein directly affect its biological function.The understanding of the conformational changes during enzyme-substrate binding will shed lights on the substrate selectivity and enzymatic mechanism of the enzyme.In this dissertation,bioinformatics methods were applied in studying the adenylate kinase(AdK)catalytic cycle and its molecular mechanism.We also combined bioinformatics with 19F NMR experimental technique to study the substrate recognition mechanism of Lecitase(?)Ultra lipase(PLA1 lipase),and the conformational change and its molecular mechanism during the combination of PLA1 and its substrate.The goal is to reveal the catalysis and substrate recognition mechanism of these two enzymes at the molecular level,and provide theoretical basis for further enzyme engineering.The work mainly includes the following two parts:1.Molecular dynamics(MD)simulation studies on the catalytic cycle of Adenylate kinase.Adenylate kinase(AdK),an important monomeric phosphotransferase in energy transfer reaction,catalyzes the reaction for which ATP transfers the terminal phosphate group to AMP to produce two ADPs.AdK undergoes a large conformational change,and forms many different binding states with different substrates.The complex structures of AdK with different substrates have been resolved,but the molecular mechanism of the catalytic process is still unclear.Here,various MD techniques,including traditional molecular dynamics simulation,enhanced sampling simulation and parallel cascade selection MD simulation,were applied to investigate different conformational states in the catalytic process of the Escherichia coli adenylate kinase(AKeco).Based on the simulation results,we analyzed the entrance/release orders of substrates during the catalytic cycle and propose a probable cycling pathway.The free AKeco tends to maintain an open conformation.ATP·Mg enters its binding pocket first,and then AMP enters,followed by the conversion of AKeco into the closed conformation.After the catalytic reaction,ADP Mg is released first,and then ADP is released and the whole catalytic cycle is completed.Contact and distance analysis between substrate and protein during this process revealed that electrostatic interaction is an important role in controlling the orders of substrate entrance and release.2.Role of C-terminal fragment of PLAT on its enzyme activity and substrate selectivity.PLA1 lipase is a commercial enzyme possessing both phospholipase and lipase activities.PLA1 lipase was generated by genetic recombination of Thermomyces lanuginosus lipase(TLL),with the C terminal fragment peptide of Fusarium oxysporum lipase(FOL).The C-terminal fragment of PLA1 was reported to be important for the phospholipase activity,but the molecular mechanism remains unclear.In this thesis,based on the PLA1 structure model obtained from homology modeling,we use molecular docking and molecular dynamics simulation to investigate the interaction between PLA1 and its different substrates.By comparing these results with those of TLL to its substrates,we revealed the conformational change of PLA1 C-terminal domain during substrate binding,and its roles in enzyme activity and substrate selectivity.Further 19F NMR experiments were carried out to verify the conformational changes of C-terminal during substrate binding.These MD and experimental results provide a basis for future studies on activity mechanism and enzyme engineering.