Dissecting the cofactor-dependent and independent bindings of PDE4 inhibitors

Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the h ydrolysis of cAMP to AMP. At the bottom of its active site lie two divalent metal ions in a binuclear motif which are involved in both cAMP binding an d catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-64 58]. Using a SPA-based equilibrium [H-3]rolipram binding assay, we have det ermined that Mg2+, Mn2+, and Co2+ all mediated a high-affinity (K-d between 3 and 8 nM) and near stoichiometric (R)-rolipram binding to PDE4. In their absence, (R)-rolipram binds stoichiometrically to the metal ion-free apoen zyme with a K-d of similar to 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficac y in catalysis, suggesting that both metal ions of the holoenzyme are invol ved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The speci fic rolipram binding to the apo-and holoenzyme is differentially displaced by cAMP, AMP, and other inhibitors, providing a robust tool to dissect the components of metal ion-dependent and independent PDE4/ligand interactions. cAMP binds to the holoenzyme with a K-s of 1.9 muM and nonproductively to the apoenzyme with a K-d of 179 muM. In comparison, AMP binds to the holo- and apoenzyme with Kd values of 7 and 11 mM, respectively. The din-finished Mg2+-dependent component of AMP binding to PDE4 suggests that most of the Mg2+/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the h ydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.