Construction Of Glutathione Peroxidase Mimics

Reactive oxygen species (ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. ROS are harmful to cells when oxidative metabolism products increase or the antioxidant defenses lack in the living organism. Lots of researches showed that ROS are related to some physiologic and pathological processes and play role in explaining the pathogenesis of many diseases. ROS are very active and very strong oxidant and they can attack biomolecules to damage the structure and function of cells and tissues.In order to scavenge ROS, the living organism has several lines of defense systems, 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 inclu- des vitamin C, vitamin E, reduced glutathione,β-carotene, metal chelate complex, calciumion antagonists, flavonoid, polyphenol compouns and antioxidant peptides, and so on. They act with antioxidant enzymes to make free radicals under a normal level of metabolism in the organism.GPx are the well-known antioxidant selenoenzymes in organisms which destroy several harmful hydroperoxides (ROOH) and then maintain the metabolic balance of ROS in vivo, thus protecting the biomembranes and other cellular components from oxidative damage. The reactions catalyzed by GPx include two substrates, thiols (RSH) and ROOH. GPx has strong antioxidant ability and it is important to treat and prevent Keshan disease, angiocardiopathy, inflammation and cancer. Due to the limitations associated with native GPx, such as instability, limited availability, big molecular weight and imm- unogenicity, many scientists have made a great deal of efforts to study the GPx mimics.Due to the lack of the GSH binding site, the early GPx mimics have generally rather low activities. Take ebselen for example, its activity only has 0.99U/μmol. It is a particularly challenging how to generate high catalytic efficiency and water soluble GPx mimics by simple chemical and biological methods. The substrate binding and the subsequent intramolecular catalysis are the two principal phenomena that underlies the activity of enzyme. When construction of enzyme model, it is necessary to consider that enzyme model can recognize and bind substrate, and that 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: specifically recognition GSH and the unique catalytic residue Sec. So we decided to construct two GPx mimics, imprinted GSH-selenosubtilisin and selenium-containing cyclodextrin, and studied their enzymic properties and biologic efficacy. We also investigated the structure of the active site of the Se-scFv-2F3 by protein engineering.1. Imprinted GSH-selenosubtilisin to mimic GPxFor imitating the active site of GPx an artificial enzyme, selenosubtilisin, was employed as a scaffold protein and imprinted using glutathione (GSH) as imprint molecule for creating specific binding sites for the substrate GSH. GSSG was first covalently linked to selenosubtilisin to form the GSH-selenosubtilisin through Se-S bond, then the GSH-selenosubtilisin was imprinted by change of pH to cast a complementary binding site for GSH recognition. The bioimprinting procedure contains partly unfolding the conformation of the selenosubtilisin and fixing the new conformation of imprinted GSH-selenosubtilisin. This bioimprinting strategy made the catalytic selenium moiety of the imprinted selenosubtilisin match well to the sulfhydryl group of GSH in GSH binding site, which facilitates the intramolecular catalysis.These imprinted GSH-selenosubtilisin exhibited remarkable enhancement of GPx activity for the reduction of H2O2 by GSH. The highest and average GPx activity of them was 597U/μmol and 462U/μmol, and it was approximately 100 times more than that for unimprinted selenosubtilisin, at the same time, it lost the ability to catalyze reduction of H2O2 by TNB reducing H2O2. This indicates that the bioimprinting successfully reconstructed 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 aromatic groups. The result demonstrated that the assumption that recognition and substrate binding are the foundation of high catalytic activity of enzyme. Compared with ebselen, a well known GPx mimic, the activity enhancement of 500-fold was observed. The second-order rate constants of the imprinted GSH-selenosubtilisin towards H2O2 is two magnitudes larger than that of the selenosubtilisin. The detailed steady-state kinetic studies showed that the new selenoenzyme followed a ping-pong mechanism for the reduction of H2O2 by GSH, and was similar to the natural occurring GPx. The reason for the high GPx activity of the imprinted GSH-selenosubtilisin is that the enzyme model can bind directly the substrate GSH, and that Sec is at an appropriate position to interact with the thiol of GSH.2. Construction of selenium-containing cyclodextrin to imitate GPx On the basis of structural understanding for GPx, we select supro- molecular host molecules, cyclodextrins, as the scaffolds of enzyme models, and introduce catalytic sites Se and cyclohexylamine near the Se sites by chemical modification. We obtain one system of GPx mimic: cyclodextrin- based GPx models, 6A,6B- cyclohexylamine- 6A',6B'- selenium- bridgedβ- cyclodextrin(6-CySeCD), which contain the substrate selectivity like natural GPx. The GPx activity of the mimic for reduction of H2O2 by glutathione is 7.9 U/μmol, which is 7.9 times of that of ebselen. The GPx activity of the mimic for reduction of H2O2 by glutathione is 1.8 times of that 6-SeCD (4.2 U/μmol). The structure of the mimic was characterized by means of laser mass spectroscopy, elemental analysis, IR and 1HMR and its selenium content and valence were determined by means of x-ray photoelectron spectra. Double reciorocal plots of the intitial velocity versus the concentration of substrates were a family of parallel lines, consistent with a Ping-Pong mechanism involving at least one covalent enzyme intermediate. Detailed steady-state kinetic studies demonstrated that the 6-CySeCD followed a ping-pong mecha- nism similar to the naturally occurring GPx.GPx plays a pivotal role in protection cells against oxidative damage. We constructed cell damage system to evaluate the biological effects of the 6-CySeCD synthesized. The experitment results showed that H2O2 can damage the cells severely and the damage was significantly reduced by 6-CySeCD through increasing the cell viability and decreasing the malondiadehyde level and the extent of DNA fragmentation. Thus, 6-CySeCD was demonstrated to be a good candidate in medicine.3. Study on the structure of the active site of Se-scFv-2F3The Se-scFv-2F3, which shows a high activity of GPx, is one of the most important enzyme mimic designed in our laboratory. But the structure of its active site have been yet unknown to us. We predicted its structure by molecular dynamics and found SerH52 and SerH59 may be the position of its catalytic group. The result of chemical modification further demonstrated the prediction is true. And now we have found two possible catalytic sites. In order to determine the accurate position of the site and find its catalytic mechanism in detail, we designed a series of experiments and prepared two mutants in which the SerH52 or SerH59 was displaced by Ala respectively. We prepared two mutants through replacing SerH52 and SerH59 by Ala respectively and wanted to identify the accurate active site of scFv through determining and comparing GPx–like activities.In conclusion mimicking the natures of molecular recognition and catalysis of enzymes by artificial enzymes is very essential for exploring the evolved biological process of enzymes as well as their relationship between structures and functions.