Volume 272, Number 18, Issue of May 2, 1997 pp. 11686-11689
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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The  Subunit of Rod cGMP-Phosphodiesterase Blocks the Enzyme Catalytic Site

(Received for publication, January 16, 1997, and in revised form, March 3, 1997)

From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

Cyclic GMP phosphodiesterase (PDE) is the effector enzyme in the visual transduction cascade of vertebrate photoreceptor cells. In the dark, the activity of the enzyme catalytic  and  subunits (P) is inhibited by two  subunits (P). Previous results have established that approximately 5-7 C-terminal residues of P comprise the inhibitory domain. To study the interaction between the P C-terminal region and P, the P mutant (Cys⁶⁸  Ser, and the last 4 C-terminal residues replaced with cysteine, P-1-83Cys) was labeled with the fluorescent probe 3-(bromoacetyl)-7-diethylaminocoumarin (BC) at the cysteine residue (P-1-83BC). P-1-83BC was a more potent inhibitor of PDE activity than the unlabeled mutant, suggesting that the fluorescent probe in part substitutes for the P C terminus in PDE inhibition. HoloPDE (P₂) had no effect on the P-1-83BC fluorescence, but the addition of P to P-1-83BC resulted in an approximately 8-fold maximal fluorescence increase. A K_d for the P-1-83BC-P interaction was 4.0 ± 0.5 nM. Zaprinast, a specific competitive inhibitor of PDE, effectively displaced the P-1-83BC C terminus from its binding site on P (IC₅₀ = 0.9 µM). cGMP and its analogs, 8-Br-cGMP and 2-butyryl-cGMP, also competed with the P-1-83BC C terminus for binding to P. Our results provide new insight into the mechanism of PDE inhibition by showing that P blocks the binding of cGMP to the PDE catalytic site.

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