Molecular Dynamics Simulation On Structure And Function Of Urease And Its Inhibitors Design And Screening

Ureases are a group of enzymes widespread in nature among plants, animals, fungal, bacterial and body, which can hydrolyze urea to ammonia and carbamic acids. Ureases play a prominent role in the overall nitrogen metabolism in nature, their key function is to provide organisms with nitrogen in the form of ammonia for growth. This in turn causes a sharp increase in pH and accumulation of NHU+, which makes negative side effects in agriculture and health. For example, soil ureases resulted in unproductive loss of nitrogen by ammonia volatilization, while ammonia toxicity and alkalinity along with accumulated nitrite may induce plant damage, thereby causing severe environmental and economic problems. Bacterial ureases have been shown to be an important virulence factor in the development of many clinical conditions, which is determined for human and animal health. The thesis is about the theoretical calculation on the structural and functional effects of nickel ion on urease, their inhibitor design and screening based on the computer aided drug design, as well as the studies of interactions between Jack bean urease and inhibitors.1. There are two nickel ions in the active site of urease, which have great significance for urease. To study the role of nickel ions on urease structure and function, acetohydroxamic acid inhibited urease and apo enzyme was modeled by molecular dynamics simulation. The presence of nickel ions was a big challenge for simulation, non-bonded model was used for nickel ions provided good reproduction of the active site as indicated in the crystallized structure. The results confirmed that urease has a rigid active site. The conformation of flap was changed in the apo enzyme, the connection between the metal center and Hisα323 was proposed to be responsible for maintaining the flap conformation, there would also be another connection from Hisα323 to active site influence the Ni coordination, Hisα323 may act as a tether for this region of the flap. The binding free energy of acetohydroxamic acid to urease was estimated using the molecular MM-GBSA method. The binding free energy was primarily driven by electrostatic interaction in the presence of nickel ions.2. Three-dimensional model of Jack bean urease were constructed on the basis of the crystal structure of Bacillus Pasteurii urease using homology modeling technique. The model was similar with the crystal structure, confirmed that the model was accurate and reasonable. Molecular docking study was used to screen database against the three-dimensional structural model looking forward to the discovery of leading compounds. Docking research got some compounds with high-scoring which may inhibit Jack bean urease. The presence of amino interfered the inhibition activity test, based on the concept of chemical structural modification, acylation was performed on amino acid compounds in aqueous environment, using acetic anhydride as the acylating reagent. The reaction has simple operation, high yield and easy processing.3. Jack bean urease inhibition activity screening model was established using the indophenol method and phenol red method. Indophenol method has the advantages of high sensitivity, good repeatability. In the course of the experiment, some amino acids and ketones compounds had false positive phenomenon, needed to be excluded. Phenol red method is not prone to false positive phenomenon, but influenced by pH of measured compounds. Combining the two methods, the compounds that got from docking were tested, and some compounds showed inhibitory activity to Jack bean urease. Compound N-Acetyl-L-4-fluorophenylalanine and 2-Acetyl-y-hydroxybutyric showed good inhibitory effect with IC50 at the micromole level.4. In July of 2010, the crystal structure of Jack bean urease was obtained. Based on the structure and the active data from bioassay tests, docking and molecular dynamics simulations were performed to investigate the interactions between Jack bean urease and inhibitors. The results showed that in active site of Jack bean urease, nickel ions could have two kinds of coordination mode:both nickel ions were pentacoordinated or Nil was pentacoordinated while Ni2 was hexacoordinated. It also proved that during the docking, the selection of bridge atom for two nickel ions was very important. When the inhibitor lacked proper bridge atom for inhibitors, the hydroxyl could be introduced for docking.The information obtained from this study could help to further understand the urease catalytic hydrolysis mechanism and the urease inhibitor action modes. It also could provide instructive information for the design of urease inhibitors.