Construction Of Photo-responsive Hydrolase Model Based On Reversible Supramolecular Assembly

Enzymes often perform their biological functions in the form of complexes rather than monomers.This enables to form a multi-active site to accelerate the reaction rate for high catalytic efficiency.However,it is little known how enzymes control the cata-lytic properties by aggregation due to the transient interaction between them.Recently,one such enzyme,silicatein,was observed to have a filamentous nanostructure for hy-drolysis of silicon alcohols to achieve biomineralization in vivo.Therefore,studying the aggregation behavior of hydrolases will help us to explore the relationship between their structure and activity and reveal the principle of multi-enzyme synergistic cataly-sis.Supramolecular chemistry can serve as a novel method for the construction of arti-ficial enzymes by periodic arrangement of the key catalytic groups of natural enzymes via self-assembly.Additionally,the activity of these supramolecular enzyme mimics can be reversibly regulated by taking advantage of their stimuli-responsive properites to achieve dynamic structural transition.Supramolecular assemblies,especially short peptide assemblies,are ideal platforms for studying the synergistic catalytic process of multiple enzymes,because of their good biocompatibility and structural components similar to natural enzymes.Up to now,most supramolecular hydrolases focus on elucidating the contributions of single catalytic components and their aggregated states to enzymatic activity,while the influ-ence mechanism of assembly structure and process is still uncertain.Thus,a photore-sponsive hydrolase model was constructed based on CB[8]-mediated supramolecular host-guest complexation to clarify the relationship between structure and activity by studying the morphological transition of their assembled structure.The details are as follows:1.Construction of photoresponsive supramolecular hydrolase based on peptide supra-amphiphiles.A photoresponsive hydrolase was constructed based on CB[8]-mediated supramolecular complexation,in which peptide-modified methylviologen（MV-HGC/MV-RGC）as hydrophilic components and azobenzene-linked alkyl chains（Azo-TBA）as hydrophobic components were synthesized to form a ternary complex with CB[8].Supramolecular hydrolase V-H₁₀R₁ with the optimal activity was obtained by regulating the co-assembly molar ratio of catalytic and substrate-binding groups.Simultaneously,the photo-induced isomerization of Azo-TBA and the assem-bly/disassembly cycles of V-H₁₀R₁ were demonstrated by UV spectroscopy and dy-namic light scattering（DLS）.2.Study on the structure–activity relationship of supramolecular hydrolase model.The response and recovery time were determined to be 10 min and 30 min,respective-ly.Three important intermediate structures during dynamic morphological transition of V-H₁₀R₁ under UV/Vis irradiation were recorded,including the budding stage,defor-mation–division stage,and cleavage stage.The structure-activity relationship of su-pramolecular hydrolase was elucidated by calculating the enzymatic kinetic parameters of the intermediate structures.Meanwhile,the synergistic catalytic mechanism of V-H₁₀R₁ that contains the influence of molecular rearrangement,hydrogen-bonding inter-actions,and hydrophobic microenvironment on both k_cat and K_m values was revealed.Photoresponsive assembly/disassembly study further confirmed that V-H₁₀R₁ can re-tain 93.3%activity over 5 cycles.In addition,cell experiments have shown that the hydrolase model has excellent biocompatibility and stability for a potential application in biomedical fields.