Investigations Of Substrate Binding And Catalytic Mechanism Of Glutathione Peroxidase Mimics

Enzymes are proteins with high substrate specificities and large rateaccelerations, as evolved natural products by several billion years. Enzymescatalyze almost all chemical reactions with stereoselectivities and specificitiesunder mild conditions. The idea of the enzymatic transition state inspires thinkingabout enzymatic catalysis for decades, and now it still serves as a conceptual guidefor understanding enzymatic catalysis. Transition states are the balance point ofcatalysis. Bonds are partially made and/or broken at the transition state, and theenergy of the extended system provides near-equal probability that the systemforms products or reverts to reactants. Enzymatic catalytic sites provide dynamicelectronic environments that increase the probability to form the transition state.Alignment of reactants in the Michaelis complex and motion of the catalytic sitearchitecture are necessary to achieve the transition state. Transition state lifetimesare a fraction of a picosecond, preventing chemical equilibrium in extendedcovalent systems. In catalysis, the procedure that enzymatic catalytic sitesdelicately match with the structure of transition state, decreases the activatedenergy of high-energy transition state efficiently, and thus facilitates the catalyzedreactions by overcoming the energy barrier.Glutathione peroxidases (GPX) are the well-known antioxidant selenoenzymesin organisms which destroy several harmful hydroperoxides (ROOH) and thenmaintain the metabolic balance of reactive oxygen species (ROS) in vivo, thusprotecting the biomembranes and other cellular components from oxidativedamage. In certain pathogenic states, the production of ROS is enhanced and theexcess ROS damage various biomacromolecules including RNA, DNA, protein,sugars and lipids, and therefore results in ROS-mediated diseases. ROS-relateddiseases include reperfusion injury, inflammatory process, age-related diseases,neuronal apoptosis, cancer and cataract. Therefore, GPX can really act asantioxidant drugs. Unfortunately, scientists can not fully understand the structuresof GPX as well as its catalysis mechanism in vivo at the present time. Fabricationof GPX models offers an ideal alternative for elucidating the origin of substratebinding and catalysis of enzyme.Mimicking the natures of molecular recognition and catalysis of enzymes byartificial enzymes is very essential for exploring the evolved biological process ofenzymes as well as their properties of structures and functions. The reactionscatalyzed by GPX include two substrates, thiols (RSH) and ROOH. On the basis ofstructural understanding for GPX, we select supramolecular host molecules,including cyclodextrins and dendrimers, as the scaffolds of enzyme models, andintroduce catalytic sites Se or Te by chemical modification. Finally, we obtain twosystems of GPX mimics: cyclodextrin-and dendrimer-based GPX models, whichcontain the substrate selectivities like natural GPX. Through studying therelationships between substrate recognition and catalysis of GPX mimics, weprovide some useful information on the catalysis of enzymes. Our work not onlyperfects the development of the research areas of cyclodextrins and dendrimers,but also promotes the understanding of catalytic properties of natural enzymes.1. Substrate Binding and Catalytic Mechanism of Tellurium-ContainingCyclodextrins as GPX MimicsIn order to model the substrate selectivity of natural GPX, we take cyclodextrinas template and simultaneously introduce catalytic group Te into the primary orsecondary side of cyclodextrin to establish a series of tellurium-containingcyclodextrin GPX models. The property of Te is similar to that of Se. Moreover,cyclodextrins are the second-generation supramolecular host. Combining thecatalytic group Te and binding site cyclodextrin, we find that the GPX modelsexhibit significant substrate selectivities. Specially, GPX mimic2,2′-ditellurobis(2-deoxy-β-cyclodextrin) (2-TeCD) is the best among them. Usingglutathione (GSH) as substrate, the catalytic activity of 2-TeCD is 24-fold higherthan that of diphenyl diselenide (PhSeSePh), a small molecular GPX mimic.However, taking 3-carboxyl-4-nitrobenzenethiol (CNBSH) as substrate, thecatalytic activity of 2-TeCD is 200,000-fold higher than that of PhSeSePh.Markedly, the catalytic activity of 2-TeCD is largely increased when the substrateis preferential. Furthermore, we find that 2-TeCD catalyzes the peroxidase reaction3 times faster than the well-known semisynthetic GPX mimic selenosubtilisin.Considering the action of substrate binding in 2-TeCD catalysis, we monitor thepossible process of molecular recognition of 2-TeCD by means of UV,fluorescence spectroscopy, NMR, binding inhibit, and molecular modeling.Wedemonstrate that the hydrophobic cavity of cyclodextrin moiety plays a key rolefor catalysis, and hydrophobic interactions are the primary driving force during theinclusion complexation of 2-TeCD. At the same time, we try to trap the reactiveintermediates to clarify the catalytic mechanism of the peroxidase reaction of2-TeCD, and discover that catalytic mechanism of 2-TeCD meets ping-pongmechanism, in which three reactive intermediates, including tellurol, tellurenicacid, and tellurosulfide were found, similar to that of natural GPX.2. Molecular Recognition and Catalytic Capacity of Tellurium-ContainingCyclodextrins as GPX MimicsIt is long known that GPX shows substrate specificity, and their activities can beaffected significantly by structurally distinct substrates. Attempt to elucidate therelationships between substrate specificity and catalytic capacity oftellurium-containing cyclodextrin GPX models is carried out through establishingthree assay systems using three different thiol substrates, GSH, CNBSH, and4-nitrobenzenethiol (NBSH), in the presence of a variety of structurally distinctROOH, H2O2, tert-butyl hydroperoxide (t-BuOOH), and cumene hydroperoxide(CuOOH), as the oxidative reagent. We find that 2-TeCD shows different catalyticactivity in different assay systems, indicating that 2-TeCD has potential substrateselectivity. This is the first report on the study of the relationships betweenmolecular recognition and catalysis in small molecular GPX mimics. Acomparative study of the three assay systems reveals that the cyclodextrin moietyof 2-TeCD endows the molecule with selectivity for ROOH and thiol substrates,and hydrophobic interactions are the most important driving forces in 2-TeCDcomplexation. NBSH is the preferred thiol substrate for 2-TeCD among the chosenthiol substrates, and CuOOH is a preferential hydroperoxide substrate. Our kineticdata shows that the second-order rate constants of 2-TeCD for aromatic thiols aresimilar to some of those of natural GPX, as high as 106-7 M-1min-1. At the sametime, we observe that the structurally diverse guest molecules drastically affect themolecular recognition ability of cyclodextrins, thus leading to remarkably differentGPX activities in the above three assay systems. Our study confirms that efficientbinding of the substrate is very essential for the catalytic ability of the GPX mimic.It is quite possible that efficient substrate binding promotes the formation oftransition state in enzyme catalysis.3. Structures and Functions of Tellurium-Containing Cyclodextrins as GPXMimicsTo understand the hypostasis of structures and catalysis of natural GPX andmechanism of molecular recognition in enzymatic two-substrate reaction, wedesign and construct direct assays systems to investigate tellurium-containingcyclodextrin GPX mimics. In the biomimetic system, not only GPX mimics holdone or two binding sites of cyclodextrin moieties, but also thiol substrates used inthese direct assays can serve as reactant as well as probe of molecular recognition.Due to the binding action of cyclodextrin moieties, all of these GPX mimicsexhibit substrate selectivities. Furthermore, all GPX mimics possibly showsomewhat directions of substrate binding owing to different catalyticmicrostructures of tellurium-containing cyclodextrins. We find that, in thetwo-substrate reactions, it is essential and efficient for simultaneously binding twosubstrates to enhance the catalytic activities of GPX mimics. The process ofsubstrate binding of GPX mimics is dynamic, and the catalytic efficiencies of GPXmimics depended strongly upon the competitive recognition of both substrates forGPX mimics. While increasing the binding ability of one substrate for GPXmimics, the binding action of GPX mimics for another substrate would decrease.Interestingly, the increase of concentration of low-affinity substrate can improvethe catalytic activities of GPX mimics. The concentration compensation canefficiently modulate the substrate binding of GPX mimics and thus affect thecatalytic activities. In addition, we observe that difunctional recognitionaccelerates the substrate specificity of GPX model more remarkably. In the GPXmodel with difunctional recognition sites, both substrates still rival each other tooccupy each of binding sites. Essentially, double binding sites facilitate respectivebinding of both substrates, and furthermore lower the intensity of competitiverecognition of both substrates. The catalytic activity of GPX models could begoverned by the product disulfide generated during catalytic cycle, and theaccumulated product can seriously inhibit the catalyzed reaction. From our work,we suggest that native GPX does not potentially contain a nice binding site forproduct disulfide, and during in vivo catalysis the catalytic capacity of GPX woulddepend on the relative concentrations of reducing and oxidizing substrates. Theprinciple that the metabolic balance of ROS is partially controlled by GPX mainlyattributes to the theory of chemical reaction balance.4. Constructions of Selenium-Containing Dendrimers Mimcking GPXLike natural enzymes, the structures of dendrimers can be precisely controlled atthe molecule level, resulting in a well-defined microenvironment. We takeadvantage of the microenvironment provided by the delicate structure of thedendrimers and introduce the catalytic center into the inner core of the dendrimerto achieve ideal GPX mimics with molecular recognition ability. We havedesigned and synthesized a series of Fréchet-type poly(aryl ether) dendrimers witha diselenide core and characterized these GPX mimics by means of elementalanalysis, 1H NMR, 13C NMR, 77Se NMR, and MALDI-TOF MS. In thebenzenethiol (PhSH) assay, dendrimer-based GPX mimics demonstrategeneration-dependent GPX activities with initial reduction rates as high as 2431.20μM?min?1 for the third-generation product. The third-generation dendrimerexhibits highest GPX activity among the reported GPX models in the nonaqueousassay, and its activity is at least 1000-fold higher than that of the famous GPXmimic ebselen. The generation-dependent activities of GPX mimics are relatedwith their binding ability for substrates. As expected, we find that dendrimer-basedGPX mimics show generation-dependent binding constant for substrate PhSH.This is the first successful example of using a dendrimer as a model for a highlyefficient GPX mimic. We anticipated that this work opens a new avenue forconstruction of high-efficiency GPX mimics and understanding of biologicalfunction of GPX.5. Structures and Catalytic Mechanism of Selenium-Containing Dendrimersas GPX MimicsThe aggregation of proteins, often involving the interaction of hydrophobicpatches, is central to many important biological phenomena. For example, manyenzymes are active only in their dimeric form. In our biomimetic systems ofdendrimers-based GPX mimics, enzyme models are much more active in theiraggregation,proved by dynamic light scattering, atom force microscopy, andmolecular modeling, and the hydrophobic interactions are the major driving forcesin the self-assembly process. Due to the relative rigid structures of dendrimers, theformed aggregations potentially resemble networks with small molecular aperture,and the delicate microenvironment is helpful for the binding of substrates incatalysis. High generation dendrimer can generate nice microenvironment forcatalytic cycle. We discover that the catalytic activities of dendrimers-based GPXmimics can be governed by the polarity of solvent, for the activity changes as thepolarity of solvent changes. During the process of increasing the polarity ofsolvent, the catalytic activity of third-generation dendrimer is most remarkablyenhanced, suggesting that the micropolarity around the core selenium issignificantly reduced with the increased dendritic generation. By means of 77SeNMR technology, we demonstrate that the catalytic mechanism ofdendrimers-based GPX mimics, which includes three reactive intermediates,selenol, selenenyl sulfide, and selenenic acid, resembles that of natural GPX. Ourkinetic data shows that the second-order rate constants of third-generationdendrimer GPX mimic for thiol substrate PhSH are as high as 105 M?1min?1, onlyone to two orders of magnitude lower than that of natural GPX. This study notonly corroborates our strategy of mimicking GPX, but also provides additionalpossibility of experimental research on understanding the aggregation of proteins.