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Related Experiment Videos

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
PubMed
Summary
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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.

Related Experiment Videos

  • 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.