Title : Construction of artificial metalloenzymes by protein refolding
Abstract:
Metalloenzymes contain a metal cofactor in a protein scaffold, which are involved in many biological processes such as nitrogen fixation, respiration, hydrocarbon oxidation, and oxygenic photosynthesis. Based on the function and mechanism of natural enzymes, in 2001, we proposed three catalytic action modes that for the best one the negative charge centers seem to be the active site for reactants or products, while the positive one seems to be the active site for transition states, resulting in great decrease in the activation energy. To meet the above requirements, artificial metalloenzymes (ArMs) can be constructed by anchoring a metal complex of positive charge on the pocket of a protein which periphery has negative charges.
To find a simple and versatile method for the incorporation of metal complexes within the protein host, lessons can be learned from protein folding. The protein folding forces include hydrophobic, electrostatic, hydrogen bond, and van der Waals interactions. In the case of metalloproteins, there is an additional folding force, ligand-metal interactions. It was reported that there is a direct relationship between folding and metal coordination. Inspired by metal-induced protein folding, we report here a simple and versatile method for the incorporation of metal complexes within the protein host by protein refolding.
The as-prepared artificial hemeprotein was characterized by UV-vis, CD, and MALDI-TOF-MS, which exhibited peroxidase-like activities with an optical pH at pH 7.0 and an optical temperature at 65°C. The Michaelis-Menten constant (Km) and maximum velocity (Vm) with guaiacol for the as-prepared hemeprotein was calculated to be 0.131 mM and 0.166 mM/s, which was signi?cantly lower compared with the value of 39.7 mM and 44.4 mM/s of native horseradish peroxidase. The artificial hemeprotein had much more affinity to the guaiacol than HRP, and had lower guaiacol oxidation efficiency than HRP.
