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J. Biol. Chem., Vol. 281, Issue 9, 5553-5558, March 3, 2006

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Phosphodiesterase-5 Gln⁸¹⁷ Is Critical for cGMP, Vardenafil, or Sildenafil Affinity

ITS ORIENTATION IMPACTS cGMP BUT NOT cAMP AFFINITY^*

From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln⁷⁷⁵ constrains the orientation of the invariant Gln⁸¹⁷ side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98–102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095–13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279–286). PDE5_Q817A and PDE5_Q775A 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 the molecular mass of the mutant proteins were unchanged compared with PDE5_WT. For PDE5_Q817A, K_m for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5_Q775A, K_m for cGMP was weakened 20-fold but was unchanged for cAMP. For PDE5_Q817A, 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 that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5_Q775A. 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.

Received for publication, September 21, 2005 , and in revised form, December 30, 2005.

^* This work was supported by National Institutes of Health Grants DK40299 and DK58277 and American Heart Association Postdoctoral Fellowship 032525B. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

¹ To whom correspondence should be addressed: Dept. of Molecular Physiology & Biophysics, Light Hall Rm. 702, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. Tel.: 615-322-4383; Fax: 615-343-3794; E-mail: Sharron.francis{at}vanderbilt.edu.

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