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Conformational difference between PDE4 apoenzyme and holoenzyme.
F Laliberté1, Y Han, A Govindarajan
1Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, H9R 4P8, Canada.
Biochemistry
|June 1, 2000
Summary
Magnesium cofactor binding induces a conformational change in phosphodiesterase 4 (PDE4), enabling high-affinity cAMP interaction and catalytic activation. This cofactor-dependent shift influences inhibitor binding, impacting drug development strategies for PDE4 enzymes.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Type 4 phosphodiesterases (PDE4s) are Mg(2+)-dependent enzymes hydrolyzing cAMP to AMP.
- PDE4 exhibits two conformations with differential rolipram inhibitor binding affinities.
- These conformational states are linked to the enzyme's interaction with its Mg(2+) cofactor.
Purpose of the Study:
- To investigate the role of Mg(2+) cofactor binding in PDE4 conformation and substrate/inhibitor interactions.
- To characterize the binding affinities of various inhibitors to apoenzyme and holoenzyme forms of PDE4.
- To elucidate the mechanism of PDE4 activation by its metal cofactor.
Main Methods:
- Utilized a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay.
- Employed L-791,760, a fluorescent inhibitor, to probe enzyme-cofactor interactions.
- Measured inhibitor displacement assays to determine binding affinities to apoenzyme and holoenzyme.
Main Results:
- L-791,760 binds apoenzyme and holoenzyme with similar affinities (K(d) ≈ 30 nM).
- Inhibitors like CDP-840, SB-207499, and RP-73401 show preferential binding to the holoenzyme.
- (R)-rolipram binds holoenzyme (K(d) = 5 nM) significantly better than apoenzyme (K(d) = 300 nM).
- cAMP exhibits reduced affinity (K(d) ≈ 170 µM) and nonproductive binding to the apoenzyme compared to the holoenzyme (K(d) ≈ 2 µM).
Conclusions:
- Cofactor binding, specifically Mg(2+), induces a high-affinity conformation for cAMP interaction in PDE4.
- This cofactor-mediated conformational change is crucial for PDE4 catalytic activation.
- Differential inhibitor binding to apo- and holoenzyme forms provides insights into drug design targeting PDE4.