Zinc dependent activation of cAMP-specific phosphodiesterase (PDE4A).
Cyclic nucleotide phosphodiesterases (PDE), including PDE4A, contain two consensus sequences (HX3HX24-26E) which have been associated with Zn2+ binding and activation with other proteins. This study shows that Zn2+ activates purified recombinant human PDE4A with an EC50 of <1 microM. The EC50 for Mg2+, the generally accepted activating metal ion, is approximately 100 microM. Zn2+ concentrations higher than 5 microM are inhibitory. Mn2+, Co2+ and Ni2+ also activate PDE4A with EC50 values of approximately 2, 3 and 10 microM, respectively. PDE4A binds 65Zn2+ with a Kd of 0.4 microM and approximately 1:1 stoichiometry. Titrations of PDE4A inhibition with Mg2+ and Zn2+ as activating metal ions showed that the competitive inhibitors R-Rolipram, CDP-840, RS-14203 and KF18280 are shifted at least 10-fold to lower potency in the presence of Zn2+. The effect is likely at the site of inhibitor binding as the Km for cAMP in the presence of Mg2+ and Zn2+ is similar (1-3 microM). The Kd of [3H]-R-Rolipram for PDE4A was increased at least 30-fold from 3 nM (with Mg2+) by the presence of Zn2+. The high affinity of Zn2+ for PDE4A indicates that this metal may play a role in the regulation of PDE4A activity.
Percival
M D
MD
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
Yeh
B
B
Falgueyret
J P
JP
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
D002413:Cations, Divalent; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium; D015032:Zinc