Structural requirements of the photoreceptor phosphodiesterase γ-subunit for inhibition of rod PDE6 holoenzyme and for its activation by transducin

Abstract

The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its γ-subunit (Pγ), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pγ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pγ78-87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the PγC terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pγ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact α2 helix of Pγ stabilize binding of C-terminal Pγ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pγ molecule and tested for activation by transducin, we found that the C-terminal region (Pγ63-87) by itself could not be displaced but that transducin could relieve inhibition of certain Pγ truncation mutants. Our results are consistent with two distinct mechanisms of Pγ inhibition of PDE6.Oneinvolves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pγ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pγ to effectively relieve its inhibitory constraint. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.