Effects Of Metal Ions And Disulfide Bonds On The Activity Of Phosphodiesterase From Trimeresurus Stejnegeri Venom

Obviously different from the other known phosphodiesterases, the phosphodiesterase from Trimeresurus stejnegeri venom (TS-PDE) consists of two different chains linked with disulfide bonds and contains both endogenous Cu2+ and Zn2+. Cu2+ and Zn2+ are important for its phosphodiesterase activity. In this study, the effects of metal ions and small-molecule reductants on its structure and activity have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, fluorescence and electron paramagnetic resonance spectroscopy. The results show that TS-PDE has one class of Zn2+ binding site and two classes of Cu2+ binding site, including the high affinity activator sites and the low affinity sites. Cu2+ ions function as a switch for its phosphodiesterase activity. The catalytic activity of TS-PDE does not have an absolute requirement for Cu2+ and Zn2+. Mg2+, Mn2+, Ni2+, Co2+ and Ca2+ are all effective for its phosphodiesterase activity. TS-PDE has seven disulfide bonds and ten free cysteine residues. L-Ascorbate inhibits the phosphodiesterase activity of TS-PDE through reduction of the Cu2+, while dithiothreitol, glutathione and tris(2-carboxyethyl)phosphine inhibit the phosphodiesterase activity of TS-PDE by reducing both the Cu2+ and disulfide bonds. The catalytic activity of TS-PDE relies on its disulfide bonds and bimetallic cluster. In addition, biologically-relevant reductants, glutathione and L-ascorbate, have been found to be endogenous inhibitors to the phosphodiesterase activity of TS-PDE.