Conformational Difference between PDE4 Apoenzyme and Holoenzyme

• Published in: Biochemistry (Easton) 2000-05, Vol.39 (21), p.6449-6458
• Main Authors: Laliberté, France, Han, Yongxing, Govindarajan, Arvind, Giroux, André, Liu, Susana, Bobechko, Brian, Lario, Paula, Bartlett, Adrienne, Gorseth, Elise, Gresser, Michael, Huang, Zheng
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - chemistry; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Animals; Apoenzymes - chemistry; Apoenzymes - metabolism; Binding Sites; Cell Line; Cyclic AMP - metabolism; Cyclic Nucleotide Phosphodiesterases, Type 4; Energy Transfer; Humans; Kinetics; Magnesium - metabolism; Phosphodiesterase Inhibitors - chemistry; Phosphodiesterase Inhibitors - pharmacology; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spectrometry, Fluorescence; Spodoptera; Structure-Activity Relationship; Transfection
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi992432w

The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg2+-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 Mg2+. 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 Mg2+) with comparable affinities (K d ∼ 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 ∼ 2 μM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K d ∼ 170 μM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.