Construction And Properties Of Nano-metalloenzymes Based On ?-hairpin Self-assembly

Mimetic,or artificial enzymes are molecules or molecular aggregates that mimic the structural features of enzyme active center,such as shape,size,and microenvironment at molecular level.With the development of nanoscience and supramolecular technologies,the construction of supramolecular mimetic enzymes with specific catalytic activity has become a hotspot in both scientific research and application.Artificial peptide metalloenzymes have peptide molecules as the basic units,and the self-assembly of peptide supramolecular structure with enzymatic catalytic activity is driven by a series of non-covalent interactions synergistically.The structure and biochemical properties of peptide metalloenzyme are akin to those of natural enzymes.Furthermore,since peptide molecules are biocompatible and easy to be modified,artificial peptides metalloenzymes would be ideal candidates for artificial enzyme fabrication with specific functions.In this thesis,the?-hairpin peptides as the model of molecules were designed by changing the amino acids sequence in the molecular structure to investigate the effect of molecular structure to the selective coordination between the peptides and metal ions.The complex of?-hairpin peptide molecule and copper ions was used to construct artificial metalloenzymes to catalyze different types of oxidation reactions.A?-hairpin peptide Ac-VYVHVKVKV^DPPTKVKVHVGV-NH₂ has been designed and synthesized with a CEM peptide synthesizer.UV-Vis and CD results indicated that the peptide coordinated with Cu²⁺through two imidazole groups,a phenolic group of tyrosine side chain and a H₂O molecule.The conformation of Ac-VYVHVKVKV^DPPTKVKVHVGV-NH₂changed from random coil to?-sheet at p H 5.0 with Cu²⁺coordination.And the folded?-hairpin peptide further self-assembled nanofibers driven by hydrogen bonding interactions between amide bonds of backbones and hydrophobic interactions among the side chains.When other metal ions were added to the solution,the peptide conformation did not change except for Zn²⁺,indicating that the coordination between peptide and metal ions was selectiveThe effect of the peptide molecule structure on the coordination with metal ions was further studied.Based on the sequence of Ac-VYVHVKVKV^DPPTKVKVHVGV-NH₂,we have designed other four?-hairpin peptide molecules.The main difference were the distance of histidine and tyrosine to the V^DPPT,as well as wether histidine and tyrosine were on the hydrophobic surface of hairpin or not.Results of CD,UV-Vis and isothermal titration calorimetry(ITC)showed that the Cu²⁺coordination and assembly ability of?-hairpin peptides with His and Tyr on the hydrophilic surface were significantly stronger than those with His and Tyr on the hydrophobic surface.Moreover,the Ac-VYVHVKVKV^DPPTKVKVHVGV-NH₂had a small electrostatic repulsion due to the uncharged glycine and tyrosine on the two ends of the hairpin,and possessed the strongest coordination ability.Finally,artificial metalloenzymes have been constructed through the coordination of?-hairpin peptides,Ac-VYVHVKVKV^DPPTKVKVHVGV-NH₂,Ac-VKVKVYVHV^DPPTHV GVKVKV-NH₂ and Ac-HKYKVHVHV^DPPTHVHVKGKH-NH₂ with Cu²⁺.And the enzymatic activity have been tested.Hydrolysis of p-nitrophenyl acetate,oxidation of benzyl alcohol,HRP reaction of 3,3',5,5'-tetramethylbenzidine(TMB)with H₂O₂ were selected as the model oxidation reactions.It was found that the self-assemblies of Ac-VYVHVKVKV^DPPTKVKVHVGV-NH₂ with Cu²⁺had the best activity in catalyzing the oxidation of benzyl alcohol and the HRP reaction of TMB.We found that the assembly of copper ion-induced hairpin peptide assembly showed good catalytic effect,indicating that a new artificial metalloenzyme with good catalytic performance was successfully designed.