Received for publication November 6, 2007.
Revised January 15, 2008.
Accepted for publication January 15, 2008.

Phosphorylation Increases Affinity of the Phosphodiesterase-5 Catalytic Site for Tadalafil

Abstract

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotidedependent 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 [³H]tadalafil to PDE5 was measured. Phosphorylation increased [³H]tadalafil binding 3-fold while cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [³H]tadalafil binding, and effects were additive only at submaximum stimulation. Consistent with the increase in affinity, phosphorylation slowed the [³H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic sitespecific cGMP analog, 2'-O-anthraniloyl-cGMP, by ~3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independent 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.

Key words: cyclic GMP, phosphodiesterase, phosphodiesterase-5, phosphorylation, reciprocity, tadalafil