Design And Study Of Antioxidant Enzyme With Double Function

Reactive oxygen species (ROS) is a by-product of oxygen metabolism, it can damageprotein and nucleic acid of living organism, aiming at ROS, organisms have established aset of defense system. Under normal conditions, the production and elimination of ROS isin balance, when generated ROS could not be eliminated in time, ROS will cause theoxidative damage of the organism, this phenomenon is called oxidative stress. In addition,the accumulation of excessive ROS is related to many diseases, even cancer. Glutathioneperoxidase (GPX) as the member of antioxidant defense system, can remove hydrogenperoxide by using GSH. About natural GPX, its structure, function and catalyticmechanism has been well known, but natural GPX’s big molecular weight, will cause theimmune response, leading to the difficulty of clinical application, besides, thedisadvantage of its limited source and poor stability are brought greater difficulty for itsdevelopment and utilization, so the artificial simulation of GPX has attract broadattention.1. GPX Mimics for Scavenging·OH Free RadicalThrough computer simulation, we found that the β-cyclodextrin structure has ahydrophobic cavity that match the substrate of GPX----GSH, besides, Diego have foundcyclodextrin molecule replaced by imidazole has the ability of removing hydroxyl freeradical (·OH). Based on the above two points, we respectively introduce imidazole andGPX catalytic atoms-selenium (Se) into the2-OH of two adjacent sugar ofβ-cyclodextrin, and finally2-ImSeCD with selenium bridge structure is obtained by airoxidation. The mimic has good GPX activity, it’s catalytic mechanism is the same as thenatural GPX. In addition, the Fenton/Rh-B-induced damage system indicated;2-ImSeCDcan suppress the reduction of absorption peak of Rh-B caused by·OH produced byFenton reaction, which shows that2-ImSeCD have the ability to scavenge·OH. Fe2+/Vc-induced mitochondrial damage experiments showed that2-ImSeCD can protect themitochondria from injury of·OH and H2O2damage, compared with, when the system was given the same GPX activity of2-SeCD and2-ImSeCD,2-ImSeCD show apparentlyprotective effect, because of its double function. The research provides an importantexperimental basis for the design of multifunctional antioxidant enzymes.2. GSH imprinted selenium-containing protein to mimic GPXTaking trypsin as the skeleton, by, the active site of serine hydroxyl (-OH) of trypsin isconverted to selenyl (SeH) by chemical mutation, which was GPX’s catalyticintermediate, by introducting oxidized glutathione (GSSG) as imprinted molecules, theGSH and imprinted protein was connected through covalent bond(Se-SG), through thepartial denaturation, Se-SG bond and the structure of amino acids of trypsin interact witheach other, then selenium atom and the sulfur atom match very well by renaturating,which facilitates the intramolecular catalysis. Finally, we obtain an imprinted enzymewith catalytic group and substrate binding site. The GPX activity is about7times thanselentryspin, which realize the enrichment of enzyme substrate and prove that theidentification and combined with the substrate is necessary for high catalytic activity ofthe enzyme once again. In addition, the imprinted enzyme optimum pH is between7.0to8.0, the optimum temperature is45℃, the mechanism of catalytic mechanism isPing-Pong mechanism, which is the same as the natural GPX. The success of preparationimprinted enzyme can help us understand how enzymes develop their catalytic activitydeeply.