Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta

J Biol Chem. 2000 Jul 7;275(27):20572-7. doi: 10.1074/jbc.M000440200.


The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / chemistry*
  • Animals
  • Binding Sites
  • Cations, Divalent / pharmacology
  • Cattle
  • Chelating Agents / pharmacology
  • Cyclic Nucleotide Phosphodiesterases, Type 6
  • Enzyme Activation / drug effects
  • Enzyme Stability
  • Eye Proteins / chemistry*
  • Kinetics
  • Metalloproteins / chemistry
  • Picolinic Acids / pharmacology
  • Protein Binding
  • Retinal Rod Photoreceptor Cells / enzymology*
  • Spectrophotometry, Atomic
  • Zinc / chemistry*


  • Cations, Divalent
  • Chelating Agents
  • Eye Proteins
  • Metalloproteins
  • Picolinic Acids
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Cyclic Nucleotide Phosphodiesterases, Type 6
  • Zinc
  • dipicolinic acid