title: Dissecting the cofactor-dependent and independent bindings of PDE4 inhibitors.
abstract: Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the hydrolysis 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 and catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-6458]. Using a SPA-based equilibrium [(3)H]rolipram binding assay, we have determined that Mg(2+), Mn(2+), and Co(2+) 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 apoenzyme with a K(d) of approximately 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficacy in catalysis, suggesting that both metal ions of the holoenzyme are involved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The specific 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 microM and nonproductively to the apoenzyme with a K(d) of 179 microM. In comparison, AMP binds to the holo- and apoenzyme with K(d) values of 7 and 11 mM, respectively. The diminished Mg(2+)-dependent component of AMP binding to PDE4 suggests that most of the Mg(2+)/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the hydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutical Research, P.O. Box 1005, Pointe Claire, Dorval, Quebec H9R 4P8, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: F
authors.initials: F
authors.lastname: Laliberté
authors.forename: B
authors.initials: B
authors.lastname: Bobechko
authors.forename: A
authors.initials: A
authors.lastname: Bartlett
authors.forename: P
authors.initials: P
authors.lastname: Lario
authors.forename: E
authors.initials: E
authors.lastname: Gorseth
authors.forename: J
authors.initials: J
authors.lastname: Van Hamme
authors.forename: M J
authors.initials: MJ
authors.lastname: Gresser
authors.forename: Z
authors.initials: Z
authors.lastname: Huang
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020889:Rolipram / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D001051:Apoenzymes; D002413:Cations, Divalent; D008024:Ligands; D010446:Peptide Fragments; D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; D003035:Cobalt; D000249:Adenosine Monophosphate; D008345:Manganese; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D008274:Magnesium; D020889:Rolipram