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Coordination of divalent metal ions in the active site of poly(A)-specific ribonuclease

Yan-Guo Ren1, Leif A Kirsebom, Anders Virtanen

  • 1Department of Cell and Molecular Biology, Uppsala University, Biomedical Center Box 596, SE-751 24 Uppsala, Sweden.

The Journal of Biological Chemistry
|September 11, 2004
PubMed

Insights

Poly(A)-specific ribonuclease (PARN) activity depends on specific metal ions and substrate length. Divalent metal ions like Mn2+ and Zn2+ rescue PARN activity and influence substrate requirements, revealing insights into enzyme function.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Poly(A)-specific ribonuclease (PARN) is a crucial 3'-exoribonuclease that degrades mRNA poly(A) tails.
  • PARN belongs to the DEDD nuclease family, with four conserved residues (Asp-28, Glu-30, Asp-292, Asp-382) essential for its activity.

Purpose of the Study:

  • To investigate the coordination of catalytically important divalent metal ions within the active site of PARN.
  • To understand how metal ion identity affects PARN's substrate length requirements and catalytic mechanism.

Main Methods:

  • Site-directed mutagenesis was used to substitute conserved residues with cysteines (PARN(D28C), PARN(E30C), PARN(D292C), PARN(D382C)).
  • Enzyme activity assays were performed using various divalent metal ions (Mg2+, Mn2+, Zn2+, Co2+, Cd2+) and different substrate lengths (adenosine trinucleotide (A3), adenosine dinucleotide (A)).
  • Phosphorothioate modifications were introduced into the substrate to probe the role of phosphate oxygens in metal ion coordination.

Main Results:

  • Mutants PARN(D28C), PARN(D292C), and PARN(D382C) showed rescued activity in the presence of Mn2+, Zn2+, Co2+, or Cd2+, indicating these residues interact with essential metal ions.
  • The shortest substrate for PARN was A3 with Mg2+ or Cd2+, while Mn2+, Zn2+, Co2+ allowed hydrolysis of shorter A substrate, demonstrating metal-ion-dependent substrate length modulation.
  • Phosphorothioate modification studies suggested that the pro-R non-bridging phosphate oxygen plays a role in cleavage, likely through metal ion coordination.

Conclusions:

  • Asp-28, Asp-292, and Asp-382 are directly involved in coordinating catalytic metal ions in PARN's active site.
  • The identity of the divalent metal ion significantly influences PARN's substrate specificity and catalytic efficiency.
  • The study provides a detailed model for metal ion binding and coordination in the PARN active site, crucial for mRNA deadenylation.

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