effect of divalent cations on the catalytic activity of the human plasma 3′-exonuclease

The 3′-exonuclease from human plasma is a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9). Here, the possibility of divalent cation influence for the 3′-exonuclease activity was investigated using the phosphorothioate congener of oligonucleotide containing all phosphorothioate internucleotide linkages of the [RP]-configuration ([RP-PS]-d[T₁₂]) as the substrate for this enzyme. It was found that the 3′-exonuclease is a metalloenzyme, i.e. its phosphodiesterase activity was completely abolished at 0.8 mM concentration EDTA and, in turn, it was restored in the presence of Mg²⁺ or Mn²⁺ ions. In addition, Mg²⁺ can be replaced effectively by Ca²⁺, Mn²⁺, or Co²⁺, but not by Ni²⁺ and Cd²⁺ during the hydrolysis of the phosphorothioate substrate in human plasma. In addition, the mechanism is postulated, by which a single internucleotide phosphorothioate bond of the SP-configuration at the 3′-end of unmodified phosphodiesters (PO-oligos), or their phosporothioate analogs (PS-oligos) protects these compounds against degradation in blood.