Ae. Granovsky et al., PROBING DOMAIN FUNCTIONS OF CHIMERIC PDE6-ALPHA PDE5 CGMP-PHOSPHODIESTERASE/, The Journal of biological chemistry, 273(38), 1998, pp. 24485-24490
Chimeric cGMP phosphodiesterases (PDEs) have been constructed using co
mponents of the cGMP-binding PDE (PDE5) and cone photoreceptor phospho
diesterase (PDE6 alpha') in order to study structure and function of t
he 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 bacul
ovirus/High Five cell system. The catalytic properties of this chimera
were practically indistinguishable from those of PDE5, whereas the no
ncatalytic cGMP binding was similar to that of native purified PDE6 al
pha'. The inhibitory gamma subunit of PDE6 (P gamma) enhanced the affi
nity of cGMP binding at noncatalytic sites of native PDE6 alpha' by si
milar to 6-fold, The polycationic region of P gamma, P gamma-24-45, wa
s 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 modu
late 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 moti
fs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a
loss of the noncatalytic cGMP binding. Our data suggest that both puta
tive noncatalytic sites of PDE6 alpha' are important for binding of cG
MP, 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.