Multiple Zinc Binding Sites in Retinal Rod cGMP Phosphodiesterase, PDE6ab*

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