Construction Of Smart Artificial Antioxidant Enzymes Based On Allosteric Proteins

Reactive Oxygen Species?ROS?produced by aerobic respiration are able to cause severe damage to biomacromolecules in organisms,leading to cataract,Keshan disease,Alzheimer's disease,cardiogenic shock,atherosclerosis and many other vital issues.Glutathione peroxidase?GPx?is able to use glutathione?GSH?as a substrate to catalyze the reduction of peroxides with the catalytic center Selenocysteine?Sec?,thus protecting the macromolecules from ROS damage.In view of the fact that GPx has excellent antioxidant effect,mimicking GPx has become one of the hot spots in the field of artificial enzyme.Especially in terms of reactivity,rivaling the catalytic efficiency of natural enzymes has always been the goal pursued by scientists.From GPx models based on small molecules to the selenium-containing antibodies,semi-synthases,bio-blotting and protein-based GPx models,a large number of highly active artificial GPx mimics were constructed.These achievements are of great significance for disease prevention,anti-aging and other objectivesIn addition to a high catalytic efficiency,a strict regulatory mechanism is one of the typical characteristics of natural enzymes.Also,ROS are not always harmful.Under normal physiological conditions,they play an important role in some cellular signal transduction.Steady state of organisms is maintained by a variety of mechanisms,simply taking efficient antioxidant drugs will lead to metabolic system disorders,which is not conducive to normal metabolism.To meet the needs of physiological balance,the ideal artificial enzymes should not only have a high activity,but also property to be well regulated.Thus,they can function intelligently.In order to achieve this goal,we must first build an efficient active center according to the traditional enzyme design method,and then use the properties of various stimulus-responsive materials to add smart characteristics such as light,temperature and p H sensitivity to artificial enzymes.This strategy will be able to give artificial enzymes the intelligence of nature.With the development and application of stimulus-responsive materials,a large number of intelligent systems have been excavated.Reversible light-induced conformational change of azobenzene,thermo-sensitive polymer and assembly of supramolecular systems can be used to control enzyme regulation.Compared with synthetic molecules,the allosteric protein scaffolds not only have similar structures to natural enzymes,but also have high biocompatibility,structural regulation accuracy and adaptability,which have attracted the attention of scientists.It is possible to design a new catalytic site on non-homologous proteins with computer aided design and related theories.In this regard,we successfully constructed artificial antioxidant enzymes by using allosteric proteins as scaffolds.Taking advantage of the conformational change property of allosteric proteins,the artificial antioxidant enzymes could be regulated in this process.Under the guidance of this idea,calcium(Ca²⁺)and adenosine triphosphate?ATP?,which are closely related to intracellular oxidative stress levels,were selected as the artificial antioxidant enzyme regulatory molecules.Correspondingly,we found Recoverin and Adenylate Kinase?AKe?,which are able to bind Ca²⁺ and ATP,respectively.These two allosteric protein scaffolds were redesigned into Ca²⁺ and ATP regulated artificial antioxidant enzymes with native-level catalytic activity,showing good protection ability for mitochondria with switchable manner.These achievements provide new ideas for the study of smart enzymes.1.Construction of Ca²⁺-responsive smart artificial antioxidant enzymeFirst,we constructed Ca²⁺-responsive artificial antioxidant enzyme.From oxidative damage to cell apoptosis,Ca²⁺ mediates the cellular signaling pathways.Also,the Ca²⁺-rich position of mitochondria has one of the most serious oxidative levels.In order to function in the presence of high concentration of Ca²⁺,we selected the Ca²⁺ binding protein Recoverin as the scaffold of the artificial enzyme.When Recoverin binds to Ca²⁺,it is in a relatively relaxed conformation,and the N-terminal myristoyl group is out of the interior of Recoverin so that the hydrophobic cavity is exposed on the surface of the protein.When Recoverin does not bind to Ca²⁺,the N-terminal myristoyl group is embedded in a conservative hydrophobic cavity,then Recoverin is in a tensed conformation.After the GPx catalytic center Sec was implanted into the position 128 of Recoverin using genetic engineering techniques,the catalytic site was exposed and fully operational in the presence of Ca²⁺,but embedded in the protein and could not function in the absence of Ca²⁺.According to this design,it is possible to establish the enzymatic activity responsive to the Ca²⁺,and to do a repeated cycle of switching regulation without significant activity decay.Furthermore,in order to ensure the catalytic activity of the artificial enzyme,we referred to the natural GPx catalytic mechanism and added the substrate binding group arginine near the catalytic site,which increased the original catalytic activity by nearly six times.Finally,this artificial enzyme achieved the same order of magnitude as the activity of human plasma GPx.The computer simulation shows that the improvement of microenvironment near the catalytic center greatly improves the shape complementarity of the molecular docking and the positive potential of the protein surface,which enhances the enzyme-substrate affinity,and eventually increases the catalytic activity.2.Construction of ATP-responsive smart artificial antioxidant enzymeSecond,we constructed ATP-responsive artificial antioxidant enzyme.In the course of ischemia-reperfusion,pathological oxidative damage is often accompanied by a significant decrease in ATP concentration.Therefore,ATP concentration is negatively correlated with oxidative damage,and ATP concentration can be used as an indicator of the level of ROS.AKe acts as an ATP-sensitive protein backbone that exhibits an open conformation in the absence of ATP,and its core domain is exposed to the surface.When AKe combines with ATP,it changes into a closed state in a tensed conformation.We used the different bareness degree of the 120 position of AKe in different conformations,where we used the genetic engineering method to implant the catalytic center Sec,and obtained an artificial antioxidant enzyme which could inhibit the activity by ATP in the system.It is noteworthy that the artificial antioxidant enzymes can be rejuvenated after ATP is consumed by the hexokinase in the system.In order to study the effect of critical amino acids around the catalytic site on the stabilization of the substrate,a series of site-directed mutations were conducted to verify the mechanism of substrate binding,and it was found by experiments that the incorporation of arginine at position 137 could increase enzyme-substrate affinity,which is conducive to the improvement of catalytic activity.In the end,the catalytic activity of the ATP-responsive artificial antioxidant enzyme also reached the same level of activity of the human plasma GPx,and it was regulated by ATP concentration with high sensitivity.3.The biological effect of smart artificial antioxidant enzymesThe two smart artificial antioxidant enzymes were able to protect mitochondria against ROS damage in a controllable manner.At the subcellular level,we used Vc/Fe₂₊ free radical induced system to simulate the oxidative damage environment in the mitochondria,and the above two artificial antioxidant enzymes were added to test their effects on mitochondria.At a high level of oxidation,the mitochondria undergo a significant expansion,leading to a decrease in structural integrity and lipid peroxidation.The mitochondrial absorbance was measured at the wavelength of 520 nm.The decrease of the absorption value indicated that the turbidity of the system decreased,the mitochondrial swelling degree increased and the mitochondria gradually lost integrity.The unsaturated fatty acids in the mitochondria are oxidized to epoxides by H2O2 and undergo a series of metabolic processes to produce malondialdehyde?MDA?.MDA and thiobarbituric acid?TBA?reacted under acidic heating to produce a pink product,which showed the maximum absorbance at 532 nm.Experiments showed that both artificial enzymes could significantly inhibit the oxidation-caused mitochondrial expansion and lipid peroxidation,and different situations in the presence or absence of regulation molecules reflected a good switching effect.