H5n1 Influenza Virus Cut The Enzyme Catalyzes The Hydrolysis Of Rna Reaction Mechanism Studies

H5N1 virus is a highly pathogenic avian influenza, and has characteristics of strong communication and high mortality after infection. The virus has formed global threat to human health. The H5N1 avian influenza polymerase plays an important role in propagation of viruses. The polymerase complexes firstly catch host cell mRNA, then, the influenza avian polymerase endonuclease cuts the mRNA as virus mRNA transcription. If the endonuclease is not activite, the virus is not able to transcribe mRNA and reproduce itself. So,the study of catalytic mechanism of the influenza virus polymerase endonclease is important to guide us to design anti-influenza drug from a microscopic point of view. These are very important theoretical and practical significance.The hydrolysis reaction of RNA backbone catalyzed by the PAN subunit endonuclease of H5N1 avian influenza virus polymerase was investigated using density functional theory and Hartree-Fock by Gaussian 03 program. The studied content in the paper mainly included the choosing computational method and reaction mechanism. The structures and frequencies of the reactants, products, intermediates and the transition states along the reaction pathways had been obtained as well the potential energy surface information.The main contents and conclusions in the thesis are listed as follows:In the 1st chapter, hazardness of H5N1 virus and functions of the endonuclease in organism's propagation are described. The endonuclease crystal structure is determined by X-ray in experiments. According to the published references, possible reaction mechanisms are proposed in terms of one-metal-ion model.In the 2rd chapter, the ab initio theory, density functional theory, transition state theory and basis set are introduced; the QM/MM method regarding to the large biological systems is introduced as well.In the 3rd chapter, on the basis of the X-ray structure (PDB:3HW3),the endonuclease active site was modeled. The model consists of 84 atoms, which includes one divalent metal ion, magnesium ion. The model was optimized by different methods. The HF method is selected by comparing the optimized geometry of the model with crystal data. For comparison, we added diffuse function for hydrogen atom and heavy atom during geometrical optimizations at the HF level of theory. The calculated results indicated that the diffuse function would not improve the accuracy of calculation obviously.In the 4th chapter, the two one-metal-ion model A and B of endonuclease active site was constructed on the basis of the X-ray structures of the enzyme (PDB:3EBJ and 3HW3). Model A includes one simplified RNA chain, one Mg2+ ion that plays a catalytic role in the reaction process, five amino acid residues and three crystallographic water molecules. Model B is composed of model A and an additional water molecule. Three possible reaction pathways for the two models were explored. Each pathway involves three key steps:(1) One proton of water molecule transfers to the O(2) atom of phosphodiester and the generated nucleophile hydroxide ion attacks on the phosphorous atom of phosphodiester simultaneously. (2) The proton connected to the O(2)atom rotates to the O(1) atom side. (3) The P-O bond breaks and the proton connected to the O(2) atom transfers to the O(1) atom at the same time, thus facilitating protonation of the leaving group. In order to determine the properties for the stable structure, the reaction, products, intermediates and the transition states were optimized and frequency analysis were performed. The single point energies were calculated by the B3LYP/6-31G(d,p) method. The favorable reaction pathway was determined by comparing potential energy surfaces. The results show that the divalent magnesium metal cation plays a catalytic role in proton transfer of a water molecule, thus facilitates generation of a hydroxide ion. The relative energy of most favorable reaction pathway (R1-TS11-INT11-TS21-INT21-TS31-P1) are 0,110.40,91.58,143.02,102.04,110.82 and-12.96 kJ·mol-1. The theoretically calculated results indicate that the relative energy of the products of three reaction pathways are-12.96,-59.80 and-30.95 kJ·mol-1In the 5th chapter, the two QM/MM models that are at the stage of before and after the catalytic hydrolysis reaction by H5N1 influenza endonuclease were constructed and optimized. Then the vibrational frequencies of the models were analyzed, and IR spectra were plotted.