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

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 <1 mol of (R)-rolipram per mole of 1−564 or 81−564 protein. Titration microcalori...