Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6αβ

Abstract

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 Zn2+ in rod PDE6 and established a requirement for tightly bound Zn2+ in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn2+/catalytic subunit. PDE with only tightly bound Zn2+ and no free metal ions was inactive, but activity was fully restored by Mg2+, Mn2+, Co2+, or Zn2+, Mn2+, Co2+, and Zn2+ also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn2+ by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg2+. This activity loss was blocked almost completely by Zn2+, less potently by Co2+ and almost not at all by Mg2+, Mn2+, or Cu2+. The lost activity was restored by the addition of Zn2+, but Co2+ restored only 13% as much activity, and other metals even less. Thus tightly bound Zn2+ is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn2+ is rapidly exchanged are likely occupied by Mg2+ under physiological conditions.