Structural Requirements of the Photoreceptor Phosphodiesterase gamma-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin
The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its gamma-subunit (P gamma), 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 P gamma are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P gamma 78-87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P gamma C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P gamma 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 alpha 2 helix of P gamma stabilize binding of C-terminal P gamma peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P gamma molecule and tested for activation by transducin, we found that the C-terminal region (P gamma 63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P gamma truncation mutants. Our results are consistent with two distinct mechanisms of P gamma inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of P gamma 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 P gamma to effectively relieve its inhibitory constraint.
Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology
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Zhang, Xiu-Jun; Skiba, Nikolai P.; and Cote, Rick H., "Structural Requirements of the Photoreceptor Phosphodiesterase gamma-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin" (2010). Journal of Biological Chemistry. 95.
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.