Human Recombinant Phosphodiesterase 4B2B Binds (R)-Rolipram at a Single Site with Two Affinities

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Abstract
The interactions between (R)-rolipram and purified human recombinant low-Km, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1−564) and an N-terminal truncated protein (amino acids 81−564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152−528) lacked high-affinity (R)-rolipram binding. The 152−528 and 81−564 proteins had similar Km's and kcat/Km's and differed less than 4-fold compared with the 1−564 protein. (R)-Rolipram inhibition plots were biphasic for the 1−564 and 81−564 proteins and fit to two states, a high-affinity (Ki = 5−10 nM) state and a low-affinity (Ki = 200−400 nM) state, whereas the 152−528 protein fit to a single state (Ki = 350−400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be R)-rolipram per mole of 1−564 or 81−564 protein. Titration microcalorimetric studies revealed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-rolipram per mole of protein and a low-affinity state with a stoichiometry of 0.6 mol of (R)-rolipram per mole of protein for the 81−564 protein. A single low-affinity state with a stoichiometry of 0.9 mol of (R)-rolipram per mole of protein was seen using the 152−528 protein. The data indicate that purified HSPDE4B2B 1−564 and 81−564 proteins contain a single binding site for (R)-rolipram and suggest that the proteins exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the protein requires amino acid residues at the N-terminus (81−151) of the protein and catalytic domain (152−528), whereas the low-affinity binding state only requires residues in the catalytic domain (152−528). Phosphorylation at residues 487 and 489 of the 81−564 protein does not appear to alter the substrate kinetics or the stoichiometry and binding affinity of (R)-rolipram.