Discovery And Molecular Mechanism Of CAT-like Activity Of Tetraodon NKEF

Natural killer enhancing factor(NKEF) was first purified from human erythroid cell cytoplasm by Shau ect. in 1993. It was identified originally by its ability to augment natural killer cytotoxicity in vitro. NKEF was also found in many other species, and proved to be a highly conserved protein widely exist in prokaryotes and eukaryotes. Researchers also found that NKEF can perform an antioxidative activity, and thus be named PrxI/II. In traditional point of view, in order to perform its antioxidative activity, NKEF must be regenerated by Trx/TrxR reductive system(with NADPH) after being oxidized to form a intermolecular dimer. In this study, we compared the activity that NKEF monomer and dimer react with H2O2, and found that besides NKEF monomer, the intermolecular dimer form of NKEF can also react with H2O2 independently without the help of Trx/TrxR reductive system, providing a supplement to traditional view of NKEF antioxidate mechanism. In this study, we also precisely determined kinetic parameters (Km, Vmax, kcat) of NKEF via luminol-chemiluminescence method. We also introduced point mutation into NKEF sequence to construct a series of mutant proteins through PCR technic. By comparing the activity that every mutant protein reacts with H2O2, we revealed the molecular mechanism and key location of NKEF that perform its CAT-like antioxidative activity.