STRUCTURAL FEATURES OF THE NONCATALYTIC CGMP BINDING-SITES OF FROG PHOTORECEPTOR PHOSPHODIESTERASE USING CGMP ANALOGS

The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of ver tebrate vision. Although the mechanism of activation of PDE by transdu cin is well understood, the role of the noncatalytic cGMP binding site s located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interac tions between cGMP and the high affinity, noncatalytic cGMP binding si tes of frog photoreceptor PDE. None of the tested cGMP analogs were ab le to bind with greater affinity than cGMP itself, and the noncatalyti c sites were unable to bind cAMP. The major determinant for discrimina tion of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this regio n of the molecule plays a secondary but significant role in stabilizin g cGMP binding to PDE through hydrogen bond interactions. The unaltere d hydrogen at the C-8 position is also important for high affinity bin ding. A significant interaction between the binding pocket and the rib ose ring of cGMP occurs at the 2'-hydroxyl position. Steric constraint s were greatest in the C-8 and possibly the C-6/N-1 regions, whereas t he C-2/N-3 and C-2' regions tolerated bulky substituents better, Sever al lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE desc ribed in this study account for both the high affinity for cGMP and th e high level of discrimination of cGMP from other cyclic nucleotides a t the noncatalytic site.