Theoretical Investigations Of The Mechanisms Of Enoyl-CoA Hydratase And Ferulic Acid Decarboxylase

In the present thesis, the density functional method is employed to study the hydration reaction mechanism of Enoyl-CoA Hydratase and non-oxidative decarboxylation of Ferulic Acid Decarboxylase. The reaction mechanisms are respectively discussed in detail. The present results are expected to help biochemists understand the essence of these reactions. The thesis includes two parts as follows.-1. Theoretical Study on the Chemical Mechanism of Enoyl-CoA Hydratase and the Existence Form of its Inhibitor in Inhibitory ActionThe catalytic mechanism of the Enoyl-CoA Hydratase was investigated using quantum chemical methods with a model based on X-ray crystal structure. In our constructed active site, the residues Glu164, Glu144， Gly141and Ala98were taken into account, and Gln162, Ala173, Gly172, Gly1754were also included. Calculations provided insight on the details of hydration reaction 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 bridging water molecule to the double bond carbon. The relative energy of this step is23.4kcal/mol. In addition, we constructed a model of the active site with an inhibitor acetoacetyl-CoA based on the crystal structure. Additionally, it is found that the acetoacetyl-CoA has a keto-enol tautomerism when it acts as an inhibitor in the reaction mechanism of ECH. The present results are expected to help us understand the essence of this kind of reactions.2. The Catalytic Mechanism of Ferulic acid decarboxylase by non-oxidative decarboxylation:A Theoretical StudyFerulic acid decarboxylase (FADase) can catalyze the transformation of ferulic acid into4-vinyl guaiacol via non-oxidative decarboxylation in microorganisms. However, the catalytic mechanism of FADase still remains unknown. On the basis of the X-ray crystal structure and experimental results of the FADase, we design several active-site models and the mechanism of FADase was investigated by using the density functional theory. Based on the calculations, we propose a new mechanism in which a proton transfer from ferulic acid to Glu134takes place by means of a water molecule at first, and then undergoes a nucleophilic attack by another water molecule, finally the product is acquired by decarboxylation assisted by an Tyr27. Our calculated results indicated that residue Glul34is crucial for the reaction, that is, the unprotonated Glul34lower extremely the barrier of the rate-limiting step, but the protonated Glu134make the reaction be difficult to occur. Moreover, residue Tyr27plays the role to stabilize the substrate, and the water molecule is suggested to directly take part in the reaction during decarboxylation and assist the proton transfer.