Abstract
Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.
Highlights
High gain signal amplification, place specific requirements on PDE6
Our mutational analysis of chimeric PDE5/ PDE6␣ enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6␣) with P␥ is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met[758], Phe[777], and Phe[781]
Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis
Summary
The enzyme must have a very low basal cGMP hydrolytic rate in the dark-adapted photoreceptors and a very high catalytic rate in the transducin-activated state. This is achieved through two unique features of PDE6: the inhibitory interaction of the catalytic subunits with the ␥-subunit and an exceptionally high kcat value for cGMP hydrolysis when the inhibition is turned off. The rationale for our search of the catalytic determinants of PDE6 was based on biochemical evidence and the crystal structure of the PDE4 catalytic domain (18 –20), which suggests the critical role of the two highly conserved metal binding motifs, His-Asn-X-X-His (I) and His-Asp-X-X-His (II), in the hydrolysis of cyclic nucleotides. Resulting chimeric PDEs and corresponding mutants have been analyzed to test our hypothesis
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