Probing domain functions of chimeric PDE6alpha'/PDE5 cGMP-phosphodiesterase.

Abstract Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6α′) in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6α′/PDE5 enzyme containing the PDE6α′ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6α′. The inhibitory γ subunit of PDE6 (Pγ) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6α′ by ∼6-fold. The polycationic region of Pγ, Pγ-24–45, was mainly responsible for this effect, while the inhibitory domain of Pγ, Pγ-63–87, was ineffective. On the contrary, Pγ failed to inhibit catalytic activity of the chimeric PDE6α′/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6α′ are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn402 → Ala resulted in an approximately 10-fold increase of theK m value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6α′ may regulate catalytic properties of the enzyme.