Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta.
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.
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Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Seryshev
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Cowan
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Wensel
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D005136:Eye Proteins / Q000737:chemistry*; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D010848:Picolinic Acids / Q000494:pharmacology; D011485:Protein Binding; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D002614:Chelating Agents; D005136:Eye Proteins; D008667:Metalloproteins; D010848:Picolinic Acids; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D015032:Zinc; C004860:dipicolinic acid