Delineation of two functionally distinct γPDE binding sites on the bovine retinal cGMP phosphodiesterase by a mutant γPDE subunit

The gamma subunit of the retinal cGMP phosphodiesterase (gamma(PDE)) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alpha(Y)). In this work, we describe a full length, doubly point-mutated gamma subunit, C68S, Y84C gamma(PDE), which binds to PDE with increased affinity but has a decreased ability to inhibit the enzyme. Fluorescence studies monitoring the competition between wild-type gamma(PDE) and the C68S, Y84C gamma(PDE) mutant suggest that the mutant gamma(PDE) binds with high affinity to only half of the total sites occupied by wild-type gamma(PDE) Competition studies between wild-type gamma(PDE) and the mutant further suggest that the wild-type protein is able to fully inhibit PDE activity even when the mutant gamma(PDE) occupies its high-affinity binding site on PDE. Taken together, our findings are consistent with a model in which there are two distinguishable binding sites for gamma(PDE) on the PDE enzyme but that only one of the two sites mediates PDE inhibition.