Probing domain functions of chimeric PDE6α'/PDE5 cGMP-phosphodiesterase

Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6 alpha') in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6 alpha'/PDE5 enzyme containing the PDE6 alpha' 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 PDE6 alpha'. The inhibitory gamma subunit of PDE6 (P gamma) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6 alpha' by similar to 6-fold, The polycationic region of P gamma, P gamma-24-45, was mainly responsible for this effect, while the inhibitory domain of P gamma, P gamma-63-87, was ineffective. On the contrary, Py failed to inhibit catalytic activity of the chimeric PDE6 alpha'/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn(193) or Asn(402), 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 PDE6 alpha' 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 the K-m value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6 alpha' may regulate catalytic properties of the enzyme.