Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serv e as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight ass ociation with the inhibitory gamma -subunits (P gamma), The P gamma block i s 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 structu ral determinants for the inhibitory interaction with P gamma and the remark able cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristi cs by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE, PDE5 is insensitive to P gamma and has a more than 100-fold lower k(cat) f or cGMP hydrolysis, Our mutational analysis of chimeric PDE5/PDE6 alpha' en zymes revealed that the inhibitory interaction of cone PDE6 catalytic subun its (PDE6 alpha') with P gamma is mediated primarily by three hydrophobic r esidues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(7 81), The maximal catalytic rate of PDE5 was enhanced by at least 10-fold wi th substitutions of PDE5-specific glycine residues for the corresponding PD E5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent t o the highly conserved metal binding motif His-Asn-X-X-His, which is essent ial for cGMP hydrolysis. Our results suggest that the unique Gly residues a llow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.