Phosphodiesterase-5 GLN-817 is critical for cGMP, vardenafil, or sildenafil affinity; Its orientation impacts cGMP but not cAMP affinity

doi:10.1074/jbc.M510372200

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Papers In Press, published online ahead of print January 5, 2006
J. Biol. Chem, 10.1074/jbc.M510372200
Submitted on September 21, 2005
Revised on December 30, 2005
Accepted on January 5, 2006

Phosphodiesterase-5 GLN-817 is critical for cGMP, vardenafil, or sildenafil affinity; Its orientation impacts cGMP but not cAMP affinity

Roya Zoraghi, Jackie D. Corbin, and Sharron H. Francis

Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615

Corresponding Author: sharron.francis{at}vanderbilt.edu

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other AA and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln775 constrains orientation of the invariant Gln817 side chain that forms bidentate bonds with 5’-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) [Sung BJ et al.(2003) Nature 425, 98-102; Huai Q et al. (2004) J. Biol. Chem. 279, 13095-101; Zhang KY et al.(2004) Mol. Cell. 15, 279-86]. PDE5Q817A and PDE5Q775A were generated to test the hypotheses that Gln817 is critical for cyclic nucleotide or inhibitor affinity and that Gln775 immobilizes the Gln817 side chain to provide cGMP/cAMP selectivity. Allosteric cGMP-binding and molecular mass of the mutant proteins were unchanged compared to PDE5WT. For PDE5Q817A, Km for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5Q775A, Km for cGMP was weakened ~20-fold, but unchanged for cAMP. For PDE5Q817A, vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln817 is a major determinant of potency, especially for vardenafil, and binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5Q775A. It is concluded that Gln817 is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln775, which perhaps restricts rotation of Gln817 side chain, is critical for cGMP affinity, but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.

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