title: Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil.
abstract: Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2'-O-anthraniloyl-cGMP, by approximately 3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.
authors.affiliation: Department of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
authors.forename: Emmanuel P
authors.initials: EP
authors.lastname: Bessay
authors.forename: Mitsi A
authors.initials: MA
authors.lastname: Blount
authors.forename: Roya
authors.initials: R
authors.lastname: Zoraghi
authors.forename: Alfreda
authors.initials: A
authors.lastname: Beasley
authors.forename: Kennard A
authors.initials: KA
authors.lastname: Grimes
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
mesh_terms: D000818:Animals; D002243:Carbolines / Q000494:pharmacology*; D020134:Catalytic Domain; D002417:Cattle; D006152:Cyclic GMP / Q000494:pharmacology*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000302:isolation & purification / Q000378:metabolism; D004357:Drug Synergism; D006801:Humans; D006868:Hydrolysis; D058986:Phosphodiesterase 5 Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D000068581:Tadalafil
chemical_list: D002243:Carbolines; D058986:Phosphodiesterase 5 Inhibitors; D000068581:Tadalafil; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP