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 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.
Rocque
W J
WJ
Department of Biochemistry, Glaxo Wellcome Research Institute, Research Triangle Park, North Carolina 27709, USA.
Tian
G
G
Wiseman
J S
JS
Holmes
W D
WD
Zajac-Thompson
I
I
Willard
D H
DH
Patel
I R
IR
Wisely
G B
GB
Clay
W C
WC
Kadwell
S H
SH
Hoffman
C R
CR
Luther
M A
MA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D002500:Centrifugation, Isopycnic; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003956:Dialysis; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism*; D011760:Pyrrolidinones / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020889:Rolipram; D013997:Time Factors
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram