Conformational difference between PDE4 apoenzyme and holoenzyme.
The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.
Laliberté
F
F
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, H9R 4P8, Canada.
Han
Y
Y
Govindarajan
A
A
Giroux
A
A
Liu
S
S
Bobechko
B
B
Lario
P
P
Bartlett
A
A
Gorseth
E
E
Gresser
M
M
Huang
Z
Z
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004735:Energy Transfer; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014162:Transfection
D001051:Apoenzymes; D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D008274:Magnesium