Studies of the Molecular Mechanism of Discrimination between cGMP and cAMP in the Allosteric Sites of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)

Illarion V Turko,Sharron H Francis,Jackie D Corbin

doi:10.1074/jbc.274.41.29038

Illarion V Turko, Sharron H Francis + Show 1 more

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https://doi.org/10.1074/jbc.274.41.29038

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Journal: The Journal of biological chemistry	Publication Date: Oct 1, 1999
Citations: 21	License type: cc-by

Affiliation: Vanderbilt University

Abstract

The regulatory domain of the cGMP-binding cGMP-specific 3':5'-cyclic nucleotide phosphodiesterase (PDE5) contains two homologous segments of amino acid sequence that encode allosteric cyclic nucleotide-binding sites, referred to as site a and site b, which are highly selective for cGMP over cAMP. The possibility that the state of protonation in these sites contributes to cyclic nucleotide selectivity was investigated. The binding of cGMP or cAMP was determined using saturation and competition kinetics at pH values between 5.2 and 9.5. The total cGMP binding by PDE5 was unchanged by variation in pH, but the relative affinity for cGMP versus cAMP progressively decreased as the pH was lowered. Using site-directed mutagenesis, a conserved residue, Asp-289, in site a of PDE5 has been identified as being important for cyclic nucleotide discrimination in this site. It is proposed that deprotonation of Asp-289 enhances the number and strength of bonds formed with cGMP, while concomitantly decreasing the interactions with cAMP.

Highlights

Discrimination between cAMP and cGMP is an essential feature of most mammalian cyclic nucleotide-regulated or cyclic nucleotide-metabolizing proteins, namely the cAMP- and cGMP-dependent protein kinases, the cyclic nucleotide-gated channels, and the 3Ј:5Ј-cyclic nucleotide phosphodiesterases (PDEs).1 the degree of discrimination and the possible molecular mechanisms that are responsible for separation of cAMP and cGMP signaling pathways vary widely [1,2,3,4,5,6]
For the PDE5, sitedirected mutagenesis has identified the ionizing residues Asp289 and Asp-478 as important participants in cGMP binding [16]. If ionization of these amino acid residues is important, the interaction with cyclic nucleotides should be sensitive to changes in pH. Studies of this variable combined with site-directed mutagenesis have been used in the present work to probe the molecular mechanism for cyclic nucleotide discrimination in the allosteric high affinity binding site a of PDE5
The binding affinity for [3H]cGMP to the allosteric sites of PDE5 declined at lower pH values (Fig. 1, ϪcAMP)

Summary

Introduction

Discrimination between cAMP and cGMP is an essential feature of most mammalian cyclic nucleotide-regulated or cyclic nucleotide-metabolizing proteins, namely the cAMP- and cGMP-dependent protein kinases, the cyclic nucleotide-gated channels, and the 3Ј:5Ј-cyclic nucleotide phosphodiesterases (PDEs).1 the degree of discrimination and the possible molecular mechanisms that are responsible for separation of cAMP and cGMP signaling pathways vary widely [1,2,3,4,5,6]. Studies of this variable combined with site-directed mutagenesis have been used in the present work to probe the molecular mechanism for cyclic nucleotide discrimination in the allosteric high affinity binding site a of PDE5.

Results

Conclusion