Theoretical Studies On Structure, Function And Catalytic Reaction Mechanism For Several Important Proteins

From the nineties of the 20th century, the theoretical research of protein has become a powerful tool to provide the strong support and the accurate prediction for the biomacromolecules experiments and drug design with the development of computer science, medicine chemistry, molecular biology and computational chemistry. Thus, the theoretical research of protein becomes a well-established and important research area. In this thesis, molecular mechanics, molecular dynamics simulations and quantum chemical calculation methods were used to study the structures, properties and reaction mechanisms of seven proteins in detail and some creative results were obtained. The main results are outlined as follows:1.The Metabolism Study of CYP2C9 and CYP2C19 for GliclazideWith homology modeling techniques, a 3D-structure model of CYP2C19 was built and refined with molecular mechanics and molecular dynamics simulations. The refined model was assessed to be reasonable by Profile-3D and PROCHECK program. With the aid of the automatic molecular docking, one substrate and two inhibitors were docked to CYP2C19 by InsightII/Affinity program. The docking results, which are in well agreement with the reported results, demonstrate that the refined model of CYP2C19 is reliable. Then, with the refined model of CYP2C19 and the crystal structure of CYP2C9, the metabolisms of them for gliclazide in two different metabolic pathways were studied and the results show that both enzymes have more favorable interaction energies and stronger affinity with gliclazide in methylhydroxylation pathway than in 6β-hydroxylation pathway. It is exciting that substrate inhibition phenomenon can be found in metabolisms of CYP2C9 and CYP2C19 for gliclazide in two metabolic pathways. Gliclazide can change the conformation of the active sites and decrease obviously the affinities between gliclazide in the active site and enzymes when it is docked in the second active sites in CYP2C9 and CYP2C19. These results are in well agreement with the kinetic experimental results.2. The Inhibition effect of 20(S)-Protopanaxadiol (PPD) and Ginsenoside Rh2 for CYP2C9 and CYP3A4With the aid of the automated molecular docking, the inhibition effect of 20(S)-Protopanaxadiol (PPD) and Ginsenoside Rh2 for CYP2C9 and CYP3A4, respectively, were studied by InsightII/Affinity program and the docking complexes were analyzed by InsightII/Ludi program. The results indicate that PPD is a competitive inhibitor for CYP2C9 but a poor inhibitor for CYP3A4, Rh2 is a noncompetitive inhibitor for CYP3A4, but a poor inhibitor for CYP2C9. Hydrophobic PPD is stabilized in the center of the substrate binding regions of CYP2C9 by hydrogen bond and have strong interactions with heme and the key residues in active site which play important role for binding the substrate. Theoretical Ki value was calculated to be 26.7μM by using Ludi score 457 of CYP2C9-PPD complex. As hydrophilic Rh2 is away from the substrate binding regions of CYP3A4, it has very weak interactions with the key residues in the active site. But the docking of Rh2 makes the conformation of CYP3A4 changed, including the position of a key residue Ser119 that leads to a decrease in catalytic activity. Theoretical Ki value is 102.8μM by using Ludi score 398 of CYP3A4-Rh2 complex. The theoretical results are well agreement with the experimental results.3. Molecular docking study of the affinity of CYP2C9 and CYP2D6 for imrecoxibThe affinity of CYP2C9 and CYP2D6 for imrecoxib was studied with the aid of the automatic molecular docking. The results indicate that CYP2C9-imrecoxib complex has higher stability and stronger affinity because CYP2C9 has more favorable interaction energy (-62.72 kcal/mol) and higher Ludi score (610) with imrecoxib than CYP2D6 (-50.22 kcal/mol and 551) and this is consistent with the results of the kinetic experiments by Li et al. By analyzing the theoretical results combined with the experimental ones, we suggest that the affinity difference is caused by the difference of the structure between CYP2C9 and CYP2D6, and the most important residues for enzyme-substrate complexes, such as Phe476, Asn204, Phe100, Leu366 and Arg108 of CYP2C9 and Phe120, Glu216, and Phe483 of CYP2D6 were also identified.4. Catalytic Reaction Mechanism of Human Photoreceptor Retinol Dehydrogenase Based on the docking results of the substrate to the 3D-structure of hRDH8 which is generated by homology modeling method, three quantum chemical calculation models with different sizes were used to investigate the catalytic reaction mechanism of hRDH8 with the aid of density functional theory (DFT). The calculations indicate that hRDH8 employs a general acid/base mechanism that a proton is transferred to the keto oxygen of the substrate after the pro-S hydride of NADPH transfer to keto carbon of the substrate. The H-transfer order is converse to that in the proposed mechanism of 17?-hydroxysteroid dehydrogenase 1 which is highly related to hRDH8 sequence. Tyr155 always provides the proton to the keto oxygen of the substrate whether unprotonated Lys159 is considered or not in the calculation models. However, protonated Lys159 changes the initial mechanism and replaces Tyr155 to provide the proton to the keto oxygen of the substrate. Moreover, protonated Lys159 can also decrease very effectively the Gibbs free energy barrier to make the reaction indeed energetically feasible. The role of Lys159 in hRDH8 is different from that in 17?-hydroxysteroid dehydrogenase 1. The solvent effect calculations indicate that the reaction is more feasible energetically in the protein electrostatic environment than in the gas phase.5. Phosphonylation and Aging Mechanisms of Mipafox and ButyrylcholinesteraseIn this study, phosphonylation and aging mechanisms of butyrylcholinesterase with mipafox were theoretically studied at the B3LYP/6-311G(d, p) level of theory. The calculated results indicate that phosphonylation process employs a two-step addition-elimination mechanism. The addition step (the first step) is supposed to be the rate-limiting step because the Gibbs free energy barrier of this step is higher than that of the ensuing elimination step. Two different calculation models reveal that the catalytic triad of butyrylcholinesterase accelerates the whole phosphonylation process and plays the catalytic role in the reaction. By comparison with phosphonylation reaction of sarin and acetylcholinesterase reported by Wang et al., Both of emzymes employs the same mechanism in phosphonylation reaction with organophosphorus (OP) compounds. However, the Gibbs free energy barrier of the rate-limiting step in present study is higher than that in phosphonylation reaction of sarin and acetylcholinesterase. We think that it may come from the difference of the nucleophilic property of the oxygen atom in the catalytic serine residue involved in both reactions. The aging process employs also a two-step addition-elimination mechanism, with the addition step as the rate-limiting step. This is the same as the phosphonylation process. The solvent effects which were evaluated via CPCM model can change the stationary structures and the negative charges around some important atoms involved in both two processes. The free energy barrier of the rate-limiting step in the phosphynlation process is strongly influenced to have a big change by the solvent effects. In the aging process, the free energy barriers of two steps keep almost unchanged.6. Inhibition Mechanism of Cholinesterases by carbamateIn this study, the density functional theory at the B3LYP/6-311G (d, p) level was applied to explore inhibition mechanism between carbamate and cholinesterases. The results indicate that the inhibition reactions with and without the catalytic effect of the catalytic triad in cholinesterases employ two-step addition-elimination mechanism, which is in good agreement with the proposed mechanism by Lin et al. The solvent has strong effect on inhibition reactions and the solvent-phase reaction with the catalytic effect of the catalytic triad is close to the real reaction under biological condition.