Construction Of Light-responsive Glutathione S-transferase Via Supramolecular Switches

The catalytic function of an enzyme is often subject to strict control of feedback loops and various trigger-induced factors in life activities.Typically,enzymes that contain a LID domain can achieve reversible regulation of its catalytic function through their own allosteric structures.This regulation utilizes the “on/off” effect of the variable domain on the catalytic site,which affects the recognition between the enzyme and the substrate molecule to control the catalytic performance.Inspired by this phenomenon,a large number of artificial smart catalysts containing stimuli-responsive groups such as azobenzene,spiropyran,and diarylethylene have been developed to change the chemo-,regio-,and stereoselectivity of reactants or products,and to modulate their catalytic reaction processes.Scientists seek applications in the biomedical field,eventually achieving an approach that mimics nature's way of strict regulation.Therefore,the construction of smart enzymes with controllable properties that are similar to those of natural enzymes has become a hot area in rapid development.In recent years,studies have shown that smart enzymes based on protein scaffolds have extremely high catalytic performance and display more regulatory modes than natural enzymes,and therefore have a good research and application value.Currently,strategies of smart enzyme design are mainly divided into two categories:(1)Creating a brand-new catalytic center in natural allosteric proteins and regulating the enzyme catalysis by using the allosteric effect of the protein scaffold to reconstruct the catalytic center domain,(2)Introducing artificially synthesized stimuli-responsive small molecules or polymers to natural enzymes through site-directed modifications to mimic the regulatory function of LIDs.However,both the strategies have some limitations due to the construction difficulty and the poor control effect.Firstly,designing a new catalytic center from scratch in the allosteric protein skeleton without catalytic function requires complicated computer design,and the overall design is difficult.Secondly,the introduction of regulatory groups to the enzyme scaffold requires precise localization and the selection of modification sites is very difficult.Thirdly,the modified stimuliresponsive polymer chains to modulate enzyme activity is relatively large and it is difficult to avoid negative effects on enzyme activity.With the rise of supramolecular chemistry,due to the suitable size and dynamic reversible characteristics,supramolecule is very suitable for the construction of stimuliresponsive smart enzymes,and it has the advantages of excellent control performance and simple operation.The smart supramolecular catalysts provide the basis for the combination of natural enzymes and supramolecular switches,particularly the stimuliresponsive host-guest macrocyclic supramolecules to modulate the catalytic activity of natural enzymes,mimicking the regulation of enzymes by natural LID domains.It can effectively combine the efficient catalysis of natural enzymes with the advantages of the supramolecular control and develop new high-performance enzyme control methods.Among many stimuli-responsive supramolecular control methods,light response can be controlled remotely and respond quickly,clean without residue,and undoubtedly most suitable for designing smart enzymes.Therefore,we successfully constructed a light-responsive supramolecular switch using the host-guest interaction between cyclodextrin(CD)and maleimide-modified azobenzene(Azo-MAM).It was selectively modified in the natural enzyme skeleton and tested the switching effect of ?-CD and ?-CD on the catalytic activity under the UV/visible light environment.At the same time,we also studied the effect of different CD molecule sizes,modification sites,and thermodynamic parameters of the binding of azobenzene-modified enzyme on enzyme activity.The details are as follows: 1.Construction of light-responsive glutathione S-transferase based on the reversible supramolecular interaction between cyclodextrin and azobenzeneGlutathione S-transferase(GST)is a detoxification enzyme.The crystal structure shows that it contains a clear hydrophobic substrate-binding cavity.The catalytic site is located at the bottom of the cavity,and there are many amino acids around it through non-covalent interactions stabilizing the substrate glutathione(GSH).Based on the structural features of GST,site-directed mutagenesis was first performed on the 117 and 124 sites near the GST catalytic cavity,and the Cys residue was introduced as a site to be modified.Simultaneously,a maleimide-modified azobenzene derivative Azo-MAM was synthesized as a guest molecule,while ?-CD and ?-CD were used as host molecules,respectively.We used a "Click" reaction to covalently link the supramolecular switch to the cysteine:(1)the trans-Azo-MAM-CD supramolecular switch was modified only at position 124;(2)the trans-Azo-MAM-CD supramolecular switch was modified at both position 117 and 124.Since ?-CD and ?-CD are selective for cis-trans azobenzenes,UV/visible light can be used to trigger the cis-trans isomerism of Azo molecules on GST surface to regulate dissociation and recombination of CD and Azo.The “opening” and “closing” of the GST substrate binding cavity are controlled to achieve reversible modulation of the GST catalytic activity.2.Study on the properties of light-responsive glutathione S-transferaseEnzymology tests of ?-CD and ?-CD to regulate GST enzyme activity.The activities of single and double site modified GST enzymes Azo-MAM-GST/K124 C and Azo-MAM-GST/L117C/K124 C were studied after 5 cycles of UV/Visible radiation,and the closing efficiency of the two supramolecular switches was calculated.Combining the relative sizes of ?-CD,?-CD and GST and the reversible control effect on the catalytic activity of the enzyme,the effect of the control size of the supramolecular switch on the regulation of enzyme activity was analyzed in detail.In addition,by measuring the second-order reaction rate constant of optically controlled GST,the thermodynamic constant of ?-/?-CD binding,and the thermodynamic constant of the binding between substrate GSH and optically controlled GST,this supramolecular strategy was verified from both kinetic and thermodynamic viewpoints.This work provides a simple way of controlling enzyme activity at the single molecular level and achieves effective regulation of biochemical reactions.