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Characterization of a Catalytic Ligand Bridging Metal Ions in Phosphodiesterases 4 and 5 by Molecular Dynamics Simulations and Hybrid Quantum Mechanical/Molecular Mechanical Calculations

Ying Xiong ^* , Hai-Ting Lu ^* , Yongjian Li ^*, Guang-Fu Yang ^*  and Chang-Guo Zhan 

^* Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, People's Republic of China; and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536

Correspondence: Address reprint requests to Chang-Guo Zhan or Guang-Fu Yang, Dept. of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 725 Rose St. COP No. 501B, Lexington, KY 40536. Tel.: 859-323-3943; Fax: 859-323-3575; E-mail: zhan{at}uky.edu.

Cyclic nucleotide phosphodiesterases (PDEs) constitute a large superfamily of enzymes regulating concentrations of intracellular second messengers cAMP and cGMP through PDE-catalyzed hydrolysis. Although three-dimensional x-ray crystal structures of PDE4 and PDE5 have been reported, it is uncertain whether a critical, second bridging ligand (BL2) in the active site is H₂O or HO^– because hydrogen atoms cannot be determined by x-ray diffraction. The identity of BL2 is theoretically determined by performing molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations, for the first time, on the protein structures resolved by x-ray diffraction. The computational results confirm our previous suggestion, which was based on QM calculations on a simplified active site model, that BL2 in PDE4 should be HO^–, rather than H₂O, serving as the nucleophile to initialize the catalytic hydrolysis of cAMP. The molecular dynamics simulations and QM/MM calculations on PDE5 demonstrate for the first time that the BL2 in PDE5 should also be HO^– rather than H₂O as proposed in recently published reports on the x-ray crystal structures, which serves as the nucleophile to initialize the PDE5-catalyzed hydrolysis of cGMP. These fundamental structural insights provide a rational basis for future structure-based drug design targeting PDEs.