Regulation of photoreceptor phosphodiesterase catalysis by its non-catalytic cGMP-binding sites

The photoreceptor 3':5'-cyclic nucleotide phosphodiesterase (PDE) is the ce ntral enzyme of visual excitation in rod photoreceptors. The hydrolytic act ivity of PDE is precisely regulated by its inhibitory gamma subunit (P gamm a), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous P gamma, as well as by syntheti c peptides corresponding to its central and C-terminal domains, to determin e whether the non-catalytic cGMP-binding sites on the catalytic alpha beta dimer of PDE allosterically regulate PDE activity. We found that the appare nt binding affinity of P gamma for PDE was 28 pM when cGMP occupied the non -catalytic sites, whereas P gamma had an apparent affinity only 1/16 of thi s when the sites were empty. The elevated basal activity of PDE with empty noncatalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE c atalytic dimer mediate this effect. No evidence for a direct allosteric eff ect of the non-catalytic sites on catalysis could be detected for the activ ated enzyme lacking bound P gamma. The intrinsic affinity of a synthetic C- terminal (residues 63-87) P gamma peptide to bind and to inhibit the hydrol ytic activity of activated PDE was enhanced 300-fold in the presence of cGM P compared with cAMP. We conclude that the binding of cGMP to the non-catal ytic sites of PDE induces an allosteric change in the structure of the cata lytic domain that greatly enhances the interaction of the C-terminus of P g amma with the catalytic domain.