Theoretical Investigations Of The Mechanisms Of Hydroxylamine And Amides Catalyzed By AmiF Formamidase Of Helicobacter Pylori

In the present thesis, the density functional method (B3LYP) is emplyed to study the Mechanism of AmiF Formamidase of Helicobacter pylori, and Deacylation Mechanism of AmiF Formamidase of Helicobacter pylori. The mechanisms of AmiF Formamidase of Helicobacter pylori reactions are discussed in detail. The present results are expected to help Biochemists understand the essence of this kind of reactions.1. The Deacylation Mechanism of hydroxymine catalyzed by AmiF Formamidase of Helicobacter pylori:A Theoretical StudyThe catalytic mechanism of the AmiF formamidase of Helicobacter pylori was investigated using quantum chemical methods with a model based on X-ray crystal structure of Amif. In our constructed active site, the residues Glu60, Glu141, His167, Lys133, Cys166were taken into account. Calculations provided insight on the details of deacylation mechanism and explained crucial role played by these residues in the reaction. The results indicate that in the gas phase and the liquid phase, rate-determining step is the proton transferring from the amino-group to Glu60. The relative energy of this step is7.01kcal/mol. The present results are expected to help us understand the essence of this kind of reactions.2. The Catalytic Mechanism of AmiF Formamidase of Helicobacter pylori: A Theoretical StudyThe catalytic mechanism of the AmiF formamidase of Helicobacter pylori was investigated using quantum chemical methods with a large model based on X-ray crystal structure of Amif. In our constructed active site, the residues Glu60, Glu141and His167were also taken into account except for Lys133and Cys166. Calculations provided insight on the details of reaction mechanism and explained crucial role played by Glu60, Glu141and His167in the reaction. Glu60was found not only to act as a general base to adopt a proton from Cys166during the nucleophilic attack and then as a general acid to protonate the amino group during the cleavage of the C-N bond but also to activate water in the decylation. Glul41forms hydrogen bond with substrate to stabilize and orient it. For the acetylation, we proposed a new stepwise mechanism. The thiol group firstly attacks to the carbon atom of formamide and produces tetrahedral intermediate that is different from previous works. Particularly, when breaking down the intermediate, a proton-transfer step takes place before the dissociation of C-N bond. In deacylation, Glu60activates the water molecule to perform the nucleophilic attack on acyl-complex and then forms an intermediate which is distinct from the usually suggested mechanism. In addition, we found His167, Lysl33, and peptide H atom of His167(peptide H) forms oxy-anion hole which stabilizes the transition state and intermediate in the whole mechanism and reduces the activation energy barriers.