Theoretical Study On The Catalytic Mechanisms Of Three Degrading Enzymes

Almost all metabolic process in the cell need enzymes in order to occur at rates fast enough to sustain life, therefore, enzyme is the material basis for life. Since enzymatic reactions have many advantages, such as specificity and fast efficiency at physiological temperature and pressure, enzymes are widely used in the industry and agriculture, as well as in our daily life. A comprehensive understanding of the catalytic mechanism would be important for the better use of enzyme. However, as the enzymatic cycle proceeds so fast, it is hard to detect the reactive intermediates, thereby the detail of the reaction mechanism cannot be obtained through experimental work. In recent years, major advances have been made in computational hardware and software and now large biochemical systems can be investigated via accurate computational techniques. One such technique is the quantum mechanics/molecular mechanics (QM/MM) technique, which simulating reactions in realistic environments. In this work, based on the crystal structures characterized by experiments, we have performed QM/MM calculations on the catalytic mechanisms in several degradation enzymes. Calculations revealed the catalytic mechanism, the states of species involved in the reaction, the detail of each elementary step and the roles of some key residues. These results provide much information for the experimental workers and also give guideline for study of relevant enzymes. The main contents as follows:(1) The catalytic mechanism of N-acyl-homoserine lactonase (AidH).A QM/MM approach is used to study the detailed catalytic mechanism of AidH using N-hexanoyl homoserine lactone (C6-HSL) as the substrate. The calculated results support that the ring-opening step is a direct ester C-0 bond cleavage rather than a step-by-step cleavage. Additionally, the electrostatic influences of eleven surrounding residues were also reported for future mutation studies. Our results may provide useful information for the development of novel treatments for plant and animal infection that relay on AHL signaling.(2) The catalytic mechanism of 2-pyrone-4,6-dicarboxylate lactonase (LigI).A QM/MM approach was employed to study the reaction mechanism of LigI. During the process, there has two intramolecular proton transfer pathways, due to which, two final hydrolysis products can be obtained. Although His31, His33 and His 180 do not directly participate in the chemical process, they play assistant roles by forming electrostatic interactions with the substrate and its involved species in activating the carbonyl group of the substrate and stabilizing the intermediates and transition states.(3) The catalytic mechanism of 2,4’-dihydroxyacetophenone dioxygenase (DAD). 2,4’-dihydroxyacetophenone dioxygenase(DAD), an iron-containing enzyme responsible for aliphatic C-C bond cleavage, catalyzes the conversion of 2,4’-dihydroxyacetophenone, a breakdown product of lignin, to 4-hydroxybenzoic acid and formic acid with concomitant consumption of molecular oxygen. To elucidate the detail, we have performed QM/MM calculations on the catalytic mechanism in DAD. Based on our calculation and the experimentally mechanism, we revised the catalytic mechanism in DAD. The revised mechanism contains eight elementary steps, in which the former two steps are as same to the previously suggested mechanism and the last step is the rate-determining step. Furthermore, calculations predict that an iron(Ⅲ)-superoxo radical complex is the reactive oxygen species, which first undergoes a triplet-quintet crossing to initiate the reaction and subsequent transformations mainly on the quintet surface. Our results provide insight into novel mechanistic pathways for other dioxygenase, especially in the context of aliphatic C-C bond cleavage.