Generation Of GPx Mimics Employed Semisynthetic Methods

Reactive oxygen species (ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states, the production of ROS is enhanced and the excess ROS damage biomacromolecules such as RNA, DNA, protein, sugars and lipids, therefore this results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, age-related diseases, neuronal apoptosis, cancer and cataract. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. GPx plays a pivotal role in antioxidant defense system of enzymatic action. Due to the limitations associated with native GPx (solution instability, limited cellularaccessibility, immunogenicity, short half-lives, costs of production, and proteolytic digestion), scientists have made a great deal of efforts to study the structure-activity correlation of native GPx, they prepared many GPx mimics and studied the biological effects of GPx mimics. But the only consideration of chemical property without the consideration of recognition and binding substrate in enzyme reaction process makes these mimics designed and synthesized have very low GPx activity. The well-known GPx mimic Ebselen, which has been undergoing III phase clinical trial as antioxidant in Japan, but its GPx activity is only about 1 U/μmol.It is a particular challenge how to generate high catalytic efficiency and water solubility GPx mimics by simple chemical and biological methods. The two principal phenomena that underlies the activity of enzyme is substrate binding and the subsequent intracomplex catalysis. When constructing an enzyme model, it is necessary to consider that enzyme model can recognize and bind substrate, and the catalytic group is at a correct position to interact with the reactive group of substrate in order to promote catalysis. The active sites of GPx are found in flat depressions on the molecular surface. Exposure of the catalytically active Sec at the binding site is consistent with the easy access of the substrates. GPx has higher substrate selectivity for GSH than that for hydroperoxides. There are two properties of GPx: specific recognition of GSH and the unique catalytic residue Sec. In order to obtain a highly efficient GPx mimic, we propose that enzyme model must recognize and bind GSH, and the Sec is at an appropriate position to interact with the thiol of GSH.Based on the structure-function correlation of native GPx, we producesemisynthetic GPx mimics, which exhibit high GPx activity.1. GSH imprinted selenium-containing protein to mimic GPxTo elucidate the catalysis mechanism and obtain enzyme for industrial use, redesign of the natural enzyme is very important .Because it is extremely difficult for heterogenous expression of selenium-containing protein, how to generate semisynthetic selenium-containing enzyme with high catalytic efficiency and substrate selectivity becomes a great challenge for scientists. In order to attain this goal, we make use of the subtilisin Carlsberg scaffolds, converting it to selenium-containing subtilisin by chemical modification, and then prepare the bioimprinted selenium-containing enzyme with GSH binding site by reconstructing the active site. Our strategy is different from the bioimprinting methods before as follow: We use covalent bond (Se-S bond) connecting the template molecule GSH and the imprinted protein to form GS-selenosubtilisin. Furthermore, by partial denaturation and renaturation, we creat the bioimprinted selenium-containing enzyme with GSH binding site. Meanwhile, this makes the selenium atom of the enzyme and the sulfur atom of the bound substrate GSH match very well, which facilitates the intramolecular catalysis. When catalyzing the reduction of H2O2 using GSH as the reducing substrate, the bioimprinted enzyme increase the activity more than 100 times compared with selenosubtilisin, at the same time, it lost the ability to catalyze TNB reducing H2O2. This indicates that the bioimprinting successfully reconstruct the binding site of selenosubtilisin, which achieve the conversion of substrate specificity, as a result, the active site of the enzyme can recognize GSH better with the cost of losing binding specificity for aromaticgroups. The GPx activity of the bioimprinted selenium-containing enzyme is 600 times higher than the well-known GPx mimic Ebselen, which re-verifies the assumption that recognition and substrate binding are the foundation of high catalytic activity of enzyme. Normally, the hydrophilic groups are exposed on the surface of the folded protein, with the hydrophobic groups hiding inside. However, there are many hydrophilic groups here in GSH, so when the partially denatured GS-selenosubtilisin refolds, owing to the interaction of the hydrophilic groups and water molecules, GSH molecule is sure to be located at the flat depression of the folded protein. Accordingly, the selenium atom is pulled to the flat depression. In the catalytic process, GSH attack the selenium-sulfur compound to form disulfide, and this makes the direct exposure of selenium atom to H2O2, so the second-order rate constants of the bioimprinted enzyme towards H2O2 is two magnitudes larger than that of the selenosubtilisin. The mechanism of the bioimprinted enzyme is Ping-Pong mechanism, similar to the natural GPx. The novel bioimprinted enzyme successfully mimics GPx, which gives us better understanding of the mechanism of enzyme.2. Tellurosubtilisin to mimic GPxTo mimic the enzyme behavior of GPx, based on the cooperation effect of the groups in the catalytic active center, scientists have synthesized a great deal of small molecule organoselenium compound GPx mimics using chemical /biological methods and principle, including the well-known Ebselen, Spector, Tornado and Mugesh catalyst. Meanwhile, the further researches of the scientists find that the organotellurium compounds also exhibit excellent GPx activities. In the GPx'scatalytic cycle, the easy redox of selenium is one of the important reasons of the high catalytic effect for reducing hydroperoxides. Tellurium and selenium are in the same family, so the redox character of them is similar, and the tellurol can decompose hydroperoxides more easily than selenol. But the instability of the organotellurium compounds results in the limited research of aryl telluride and dialkyl telluride, which can not exhibit the GPx activity of the tellurium well. In order to have a platform to study the activity of tellurium, we use the subtilisin Carlsberg as protein scaffolds, selectively activate the hydroxyl of serine in the binding site using the protease inhibitor PMSF, and then make the nucleophilic substitution with NaHTe. In this way serine is converted to tellurocysteine, so we get tellurosubtilisin from subtilisin. Hilvert and coworkers have prepared selenosubtilisin with similar method, which stabilizes selenium by the interaction with the amino acid residues in the active site of subtilisin. Given the similarity in chemistry between selenium and tellurium, we presume the active site of subtilisin can have the same effect to stabilize tellurium as well. Tellurosubtilisin retains the activity as before even after four months when preserved in 4°C or after hundreds of catalytic cycles. All these results of the experiments verified our presumption that the active site of subtilisin can stabilize tellurium-containing prosthetic group. As GPx mimics, when catalyzing the reduction of hydroperoxides with aryl thiol TNB as the reducing substrate, tellurosubtilisin is at least 20 000 times more efficient than diphenyl diselenide, a well-studied antioxidant. Double-reciprocal plots of initial rate versus substrate concentration yields a series of linear plots that all intersect at a point in the third quadrant, and it fits the sequential kinetics reaction well. This indicates the formation of a ternary complex between enzyme,thiol, and hydroperoxide prior to product release. The detailed kinetic studies show that the tellurosubtilisin reserves the substrate specificity of subtilisin for aryl group, and in catalytic reduction of hydroperoxides by NTP the bimolecular reaction rate constants between tellurosubtilisin and NTP depend on the intrinsic reactivity of hydroperoxides.