Delineation of Two Functionally Distinct γPDE Binding Sites on the Bovine Retinal cGMP Phosphodiesterase by a Mutant γPDE Subunit

The γ subunit of the retinal cGMP phosphodiesterase (γPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the α subunit of the GTP-binding protein transducin (αT). In this work, we describe a full length, doubly point-mutated γ subunit, C68S, Y84C γPDE, which binds to PDE with increased affinity but has a decreased ability to inhibit the enzyme. Fluorescence studies monitoring the competition between wild-type γPDE and the C68S, Y84C γPDE mutant suggest that the mutant γPDE binds with high affinity to only half of the total sites occupied by wild-type γPDE. Competition studies between wild-type γPDE and the mutant further suggest that the wild-type protein is able to fully inhibit PDE activity even when the mutant γPDE 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 γPDE on the PDE enzyme but that only one of the two sites mediates PDE inhibition.