Critical Amino Acids in Phosphodiesterase-5 Catalytic Site That Provide for High-Affinity Interaction with Cyclic Guanosine Monophosphate and Inhibitors
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- 作者：Roya Zoraghi ; Sharron H. Francis ; Jackie D. Corbin
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：November 27, 2007
- 年：2007
- 卷：46
- 期：47
- 页码：13554 - 13563
- 全文大小：380K
- 年卷期：v.46,no.47(November 27, 2007)
- ISSN：1520-4995
文摘
The molecular bases for phosphodiesterase 5 (PDE5) catalytic-site affinity for cyclic guanosinemonophosphate (cGMP) and potency of inhibitors are poorly understood. Cocrystal structures of PDE5catalytic (C) domain with inhibitors reveal a hydrogen bond and hydrophobic interactions with Tyr-612,hydrogen bonds with Gln-817, a hydrophobic clamp formed by Phe-820 and Val-782, and contacts withHis-613, Leu-765, and Phe-786 [Sung et al. (2003) Nature 425, 98-102; Huai et al. (2004) J. Biol.Chem. 279, 13095-13101]. Present results of point mutations of full-length PDE5 showed that maximumcatalysis was decreased 2650-fold in H613A and 55-fold in F820A. Catalytic-site affinities for cGMP,vardenafil, sildenafil, tadalafil, or 3-isobutyl-1-methylxanthine (IBMX) were respectively weakened 14-,123-, 30-, 51-, and 43-fold for Y612A; 63-, 511-, 43-, 95- and 61-fold for Q817A; and 59-, 448-, 71-,137-, and 93-fold for F820A. The data indicate that these three amino acids are major determinants ofaffinity for cGMP and potency of selective and nonselective inhibitors, and that higher vardenafil potencyover sildenafil and tadalafil results from stronger contacts with Tyr-612, Gln-817, and Phe-820. Affinityof V782A for cGMP, vardenafil, sildenafil, tadalafil, or IBMX was reduced 5.5-, 23-, 10-, 3-, and 12-fold, respectively. Change in affinity for cGMP, vardenafil, sildenafil, or IBMX in Y612F, H613A, L765A,or F786A was less, but affinity of H613A or F786A for tadalafil was weakened 37- and 17-fold,respectively. The results quantify the role of PDE5 catalytic-site residues for cGMP and inhibitors, indicatethat Tyr-612, Gln-817, and Phe-820 are the most important cGMP or inhibitor contacts studied, and identifyresidues that contribute to selectivity among different classes of inhibitors.