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 vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE. None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination 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 region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2'-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2' regions tolerated bulky substituents better. Several 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 described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.
Hebert
M C
MC
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA.
Schwede
F
F
Jastorff
B
B
Cote
R H
RH
D000818:Animals; D001001:Anura; D001665:Binding Sites / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D010786:Photoreceptor Cells / Q000201:enzymology*; D012160:Retina / Q000502:physiology
D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP