HUMAN RECOMBINANT PHOSPHODIESTERASE 4B2B BINDS (R)-ROLIPRAM AT A SINGLE-SITE WITH 2 AFFINITIES

The interactions between (R)-rolipram and purified human recombinant l ow-K-m,, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were i nvestigated using biochemical, kinetic, and biophysical approaches. Th e full-length protein (amino acids 1-564) and an N-terminal truncated protein (amino acids 81-564) exhibited high-affinity (R)-rolipram bind ing, whereas an N-terminal and C-terminal truncated protein (amino aci ds 152-528) lacked high-affinity (R)-rolipram binding. The 152-528 and 81-564 proteins had similar K-m's and k(cat)/K-m's and differed less than 4-fold compared with the 1-564 protein. (R)-Rolipram inhibition p lots were biphasic for the 1-564 and 81-564 proteins and fit to two st ates, a high-affinity (K-i = 5-10 nM) state and a low-affinity (K-i = 200-400 nM) state, whereas the 152-528 protein fit to a single state ( K-i = 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 microcalorimetric studies reveal ed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-r olipram per mole of protein and a low-affinity state with a stoichiome try of 0.6 mol of (Ri-rolipram per mole of protein for the 81-564 prot ein. 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 conta in a single binding site for (R)-rolipram and suggest that the protein s exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the prot ein requires amino acid residues at the N-terminus (81-151) of the pro tein and catalytic domain (152-528), whereas the low-affinity binding state only requires residues in the catalytic domain (152-528). Phosph orylation at residues 487 and 489 of the 81-564 protein does not appea r to alter the substrate kinetics or the stoichiometry and binding aff inity of (R)-rolipram.