The 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 22.214.171.124/EC 126.96.36.199). 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.
KeywordsThe human plasma 3′-exonuclease NPP1 Catalysis Phosphorothioates
The authors wish to thank Krzysztof Domanski for synthesis of the PS oligonucleotides 2 and 3. This study was supported by 502-17-692 project from Medical University of Łódź (to M.W.).
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